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UBC Theses and Dissertations

The clastogenic activity of phenolic oxidation products Hanham, Ann Frances 1983

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THE CLASTOGENIC ACTIVITY OF PHENOLIC OXIDATION PRODUCTS by ANN FRANCES HANHAM B . S c , U n i v e r s i t y of Toronto, 1976 M.Sc, Simon F r a s e r U n i v e r s i t y , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1983 (c)Ann Frances Hanham, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Several e p i d e m i o l o g i c a l s t u d i e s .have demonstrated the importance of d i e t i n the development of g a s t r o - i n t e s t i n a l carcinomas i n man. This study examines the r o l e of plant p h e n o l i c s , major components of the human d i e t . Employing a CHO c e l l t e s t system, i t was observed that phenolics w i t h at l e a s t two hydroxyl groups i n the ortho p o s i t i o n , r e l a t i v e to each other were p a r t i c u l a r l y c l a s t o g e n i c . This a c t i v i t y was abolished by the a d d i t i o n of S9, a r a t l i v e r microsomal p r e p a r a t i o n . The c l a s t o g e n i c a c t i v i t y of these compounds was found to increase w i t h time, a l k a l i n e pH, and the presence of t r a n s i t i o n metals. I t was t h e r e f o r e deduced that the source of a c t i v i t y might be an o x i d a t i v e by-product. High' pressure l i q u i d chromatography was used to separate out these o x i d a t i v e products. No a c t i v i t y was found to r e s i d e i n any of the separated components or combinations of components. Further study therefore centred on o x i d a t i v e products not r e t a i n e d by chromatography and on those l a b i l e to t h i s . p r o c e s s . Under o x i d a t i v e c o n d i t i o n s , the presence of hydrogen peroxide was detected. L e v e l s measured were s u f f i c i e n t to e x p l a i n the c l a s t o g e n i c a c t i v i t y of completely o x i d i z e d s o l u t i o n s of phenolic a c i d s . A d d i t i o n of the enzyme, c a t a l a s e , appeared to a b o l i s h a l l a c t i v i t y of completely o x i d i z e d s o l u t i o n s . Hydrogen peroxide could not, however, account f o r the genotoxic e f f e c t s measured i n f r e s h l y prepared s o l u t i o n s . The presence of superoxide was detected i n a c t i v e l y o x i d i z i n g s o l u t i o n s of p l a n t p h e n o l i c s . I t s production appeared to be pH-dependent. A d d i t i o n of superoxide dismutase increased the c l a s t o g e n i c a c t i v i t y of compounds t e s t e d , presumably by converting superoxide to peroxide, a more s t a b l e o x i d a t i v e by-product. i A d d i t i o n of t y r o s i n a s e , a monophenol oxidase, a l s o increased the c l a s t o g e n i c a c t i v i t y of f r e s h l y prepared s o l u t i o n s . Since t h i s enzyme ca t a l y z e s the o x i d a t i o n of s e v e r a l phenolics without subsequent generation of peroxide, i t was deduced that phenolic f r e e r a d i c a l s must al s o be present which could be at l e a s t p a r t i a l l y r e s p o n s i b l e f o r the enhanced b i o l o g i c a l a c t i v i t y . E l e c t r o n s p i n resonance proved t h i s was the case. Using e l e c t r o n s p i n resonance, the primary o x i d a t i v e products were c h a r a c t e r i z e d both at high pH and by enzymatic a c t i v a t i o n . The r e s u l t s obtained agree w i t h those published i n the l i t e r a t u r e . S everal r e p o r t s i n the l i t e r a t u r e have suggested that phenolics may a l s o act as f r e e r a d i c a l scavengers. The importance of p l a n t phenolics i n d i e t may t h e r e f o r e depend on the o x i d a t i v e c o n d i t i o n s of the system to be t e s t e d . Under o x i d a t i v e c o n d i t i o n s , free r a d i c a l s appear to be generated, which are capable of causing mutations and chromosomal rearrangements. Phenolic o x i d a t i o n products may t h e r e f o r e p l a y a r o l e as i n i t i a t o r s and promotors of c a r c i n o g e n e s i s . However, under a l t e r n a t e c o n d i t i o n s , p h e n o l i c s may a l s o act to scavenge f r e e r a d i c a l s , and could t h e r e f o r e be c l a s s e d as i n h i b i t o r s of c a r c i n o g e n e s i s . TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES •• v i i i ACKNOWLEDGEMENTS x INTRODUCTION • 1 Biological Significance of Phenolics 1 Class i f icat ion of Phenolics 4 A. Simple Phenols.. . 4 B. Phenolic Acids 5 i) Benzoic Acids 5 i i ) Cinnamic Acids 6 C. Flavonoids 8 D. Tannins 9 i) Hydrolysable Tannins 9 i i ) Condensed Tannins 10 E. Lignins 12 Testing of Phenolics 13 The CHO C e l l Chromosome Aberration Test 14 Identif ication of Hydrogen Peroxide 18 Identif ication of Superoxide 19 Measurement of Phenolic Free Radicals 20 MATERIALS AND METHODS 24 C e l l Cultures 24 Chemicals 24 iv C e l l Cultures and Assay for Chromosome Aberrations 25 Controls for CHO C e l l Conditions 26 Preparation of S9 26 Preparation of Metal Solutions 27 High Pressure Liquid Chromatography 27 Assay for Hydrogen Peroxide 28 Assay for the Presence of Superoxide 28 Electron Spin Resonance 35 RESULTS 37 The Clastogenic Act iv i ty of Plant Phenolics 37 Oxidation Effects 41 Enzymatic Oxidation 47 Chromatographic Separation 50 Oxidation Intermediates 54 i) Generation of Peroxide 54 i i ) Generation of Superoxide 57 i i i ) Generation of Phenolic Free Radicals 63 DISCUSSION 95 Phenolics and the Environment 95 The Induction of Chromosome Abnormalities by Phenolics 97 The Oxidation of Phenolics 100 The Characterization of Phenolic Oxidation Products . . . . 103 Electron Spin Resonance of Phenolic Free Radicals 113 The Importance of Phenolic Oxidative By-products 116 v Other Dietary Sources of Free Radical Oxidation Products 121 Biological Protection Mechanisms 122 Oxidation Reactions in Man 123 Phenolics in the Process of Carcinogenesis 125 Phenolics in the Ini t iat ion of Carcinogenesis 125 Phenolics as Promotors of Carcinogenesis 126 Phenolics as Inhibitors of Carcinogenesis 129 SUMMARY 130 REFERENCES 131 APPENDICES 143 I . Simple Phenols 143 II . Phenolic Acids. 144 III . Flavonoids. 145 v i LIST OF TABLES TABLE PAGE Background Variation in the CHO C e l l Test System 38 The Clastogenic Act iv i ty of Phenolics in a CHO C e l l Test System 39 The Relative Clastogenic Act iv i ty of Oxidized Solutions of Caffeic Acid 42 The Relative Clastogenic Act iv i ty of Oxidized Solutions of Chlorogenic Acid 43 Relative Clastogenic Act iv i ty of Phenolics in the Presence of Transition Metals 46 Relative Clastogenic Act iv i ty of Phenolics in the Presence of Enzymes 48 Relative Clastogenic Act iv i t ies of Caffeic Acid in the Presence of Concurrent Applications of Enzymes 49 Relative Clastogenic Act iv i ty of Autooxidized Caffeic Acid in the Presence of Catalase Enzyme 51 Hydrogen Peroxide Content of Freshly Prepared and Autooxidized Caffeic Acid 58 The Clastogenic Act iv i ty of Freshly Prepared and Autooxidized Caffeic Acid 59 The Clastogenic Act iv i ty of Commercially Available Hydrogen Peroxide 60 Proportions of Cancer Cases Attributed to Various Factors by Different Authors 96 Genotoxic Effects Measured With Plant Phenolics 98 v i i LIST OF FIGURES FIGURE PAGE CHO C e l l Aberrations 16 Hydrogen Peroxide Reference Curve 29 Superoxide Reference Curve 33 Caffeic Acid Absorbance Spectrum at Various pH Values 44 HPLC Separation of Oxidized Solutions of Caffeic Acid 52 Accumulation of 1^ 02 with Time . 55 Rate of Production of 0^ " at Various pH Values 61 Change in Absorbance in Caffeic Acid at pH 9.0 64 Change in Absorbance in Caffeic Acid with Horseradish Peroxidase 67 Change in Absorbance in G a l l i c Acid at pH 9.0 69 Change in Absorbance in Ga l l i c Acid with Horseradish Peroxidase 71 ESR Spectra of m-coumaric, p-coumaric, Feru l i c , and Protocatechuic Acids 73 ESR Spectra of Ga l l i c Acid at pH 12.0 and Crystal l ine DPPH 76 ESR Spectra of Freshly Prepared Gal l i c Acid at pH 7.75, and after 3 Days of Incubation 78 ESR Spectra of Ga l l i c Acid at pH 7.75, and in the Presence of Horseradish Peroxidase 80 ESR Spectra of Ga l l i c Acid at pH 7.75, and in the Presence of Tyrosinase 83 ESR Spectra of Crystal l ine DPPH and Caffeic Acid at pH 12.0. 85 ESR Spectrum of Caffeic Acid at pH 12.0 88 ESR Spectra of Caffeic Acid Solutions at pH 7.5, and in the Presence of Horseradish Peroxidase 90 v i i i ESR Spectra of Caffeic Acid Solutions at pH 7.5, and in the Presence of Tyrosinase 92 Resonance Stabil ization and Dimer Formation in Phenols 101 Manganese Catalysis of Peroxide Formation 106 A l k a l i Production of Superoxide Free Radicals 109 Pathways for the Production of Cel lular Damage by Superoxide Anion Free Radicals 119 ix ACKNOWLEDGEMENTS The laboratory research for the work reported in this thesis was carried out at the B.C. Cancer Research Centre. I would l ike to extend my sincerest thanks for the help and support that I received from a l l the people who work at the Centre. In particular I would l ike to express my appreciation to Dr. H. F. Stich, my research supervisor, and to Dr. B. P. Dunn, my technical supervisor. The technical assistance of Ms. Grace Wood and Mr. Paul Lam is also gratefully acknowledged. The ESR measurements reported in this thesis were carried out in the Department of Food Science, and in the Department of Chemistry at the University of Vic tor ia . I would l ike to express my appreciation to Dr. Paul West for a l l his guidance and support in analyzing this data. My friend, J u l i Brosing, was instrumental in helping me produce the f ina l copy, while writing her own thesis. F ina l ly , I would l ike to attempt to thank my family and husband for their continued support. Somehow they just seem to make i t a l l worthwhile. Financial support was provided by the National Cancer Institute through a National Cancer Institute Studentship. x INTRODUCTION In 1964, the World Health Organization r e l e a s e d a r e p o r t on the p r e v e n t i o n of cancer i n man. This r e p o r t concluded t h a t a m a j o r i t y of common f a t a l cancers were a r e s u l t of l i f e s t y l e and other environmental f a c t o r s and were t h e r e f o r e i n p r i n c i p l e , preventable. The r e p o r t was a c u l m i n a t i o n of e p i d e m i o l o g i c a l research which suggested t h a t as many as 80 to 90% of a l l human tumours could; be found i n t h i s category. The i m p l i c a t i o n s of such a statement were and are enormous. Included i n l i f e s t y l e and other environmental f a c t o r s are the components: d i e t , c u l t u r e (drug, tobacco, and a l c o h o l u s e ) , occupation, r a d i a t i o n ( s u n l i g h t ) exposure, as w e l l as a i r and water q u a l i t y e f f e c t s . Of these components, d i e t appears to have the most f a r reaching i m p l i c a t i o n s i n the p r e v e n t i o n of cancer. I t i s through d i e t t h a t man comes i n contact w i t h a m a j o r i t y of chemicals ( f o r reviews see, S t i c h e t a l . , 1981c; Hietanen, 1981; M i l l e r and M i l l e r , 1979; Sugimura, 1979; and Clayson, 1975), and i t i s through d i e t modulation t h a t the frequency o f tumours at s p e c i f i c s i t e s can be a l t e r e d . Epidemiology supports t h i s view (Hirayama, 1981; Higginson, 1979; Sugimura, 1979; Wynder and G o r i , 1977; Wynder et a l . , 1977). The purpose of t h i s study was to examine a major component of d i e t , the p l a n t p h e n o l i c s . These compounds have a unique s t r u c t u r e e n a b l i n g them to p l a y a v a r i e t y of r o l e s i n c e l l u l a r development. Because of t h e i r h i g h r e a c t i v i t y and a b i l i t y t o permeate c e l l membranes, they have been found to induce s i g n i f i c a n t t o x i c i t y i n c e l l s . This study examines some of the r e a c t i o n mechanisms i n v o l v e d i n inducing genetic damage. B i o l o g i c a l S i g n i f i c a n c e of P h e n o l i c s : P h e n o l i c compounds make up a c o n s i d e r a b l e f r a c t i o n of p l a n t m a t e r i a l consumed as food. I t has been estimated t h a t a minimum of 600 1 mg of p h e n o l i c m a t e r i a l i s ingested each day i f i the normal human d i e t (Powell et a l . , 1974). I n t e r e s t i n g l y , higher animals cannot themselves synthesize compounds w i t h benzenoid r i n g s from a l i p h a t i c p r e c u r s o r s (phenolic s t e r o i d s being one notable e x c e p t i o n ) . P l a n t s are t h e r e f o r e the source of n e a r l y a l l phenols found i n animals. Even such e s s e n t i a l phenols as t y r o s i n e , catecholamine, t h y r o x i n e , v i t a m i n E tocopherols, and v i t a m i n K menadiones are drawn d i r e c t l y or i n d i r e c t l y from p l a n t sources ( S i n g l e t o n , 1981). The r e a c t i v e nature of p h e n o l i c compounds makes them important i n t e r m e d i a r i e s i n metabolism. T h e i r a b i l i t y to p a r t i c i p a t e i n o x i d a t i o n - r e d u c t i o n r e a c t i o n s allows them to p l a y both a genotoxic and b e n e f i c i a l r o l e i n c e l l u l a r development. Phenolics form the backbone of main b i o s y n t h e t i c pathways i n aromatic amino a c i d s y n t h e s i s . They may a l s o p l a y a r o l e as i n h i b i t o r s of c a r c i n o g e n e s i s . G a l l i c and t a n n i c a c i d s , f o r example, have both been found to be e f f e c t i v e i n h i b i t o r s of n i t r o s a t i o n at g a s t r i c pH ( M i r v i s h et a l . , 1975a; 1975b). At the same time, some are potent t o x i n s capable of inducing d i e t a r y r e a c t i o n s i n animals. N a t u r a l l y o c c u r r i n g p h e n o l i c substances found t o be c a r c i n o g e n i c i n animal t e s t s i n c l u d e the potent a f l a t o x i n s ( f o r a review see M i l l e r and M i l l e r , 1979), s a f r o l e ( M i l l e r et a l . , 1979; W i s l o c k i et a l . , 1977), q u e r c e t i n (Pamukcu, 1980), and tannins (Kdrpassy, 1961). They may a l s o act as cocarcinogens. The c h l o r o g e n i c a c i d content of c o f f e e , f o r example, i s s u f f i c i e n t to c a t a l y z e formation of N-nitrosamine carcinogens from n i t r a t e and secondary amines ( C h a l l i s and B a r t l e t t , 1975; Mori and Hirono, 1977). The f u n c t i o n of phenols i n p l a n t s i s unknown. One hypothesis i s t o confer p r o t e c t i o n of p l a n t s from diseases caused by b a c t e r i a , f u n g i , and v i r u s e s . Reasons f o r t h i s assumption i n c l u d e the f a c t t h a t many p h e n o l i c compounds have a n t i - m i c r o b i a l a c t i v i t y , p h e n o l i c s are w i d e l y d i s t r i b u t e d 2 in plants, and that there are often large ""increases in phenolic synthesis in plants after infection with plant pathogens (Friend, 1981). In animals, phenylalanine (a-aminohydrocinnamic acid) and i t s metabolite tyrosine (a-amino-p-hydroxyhydrocinnamic acid) are important amino acids required for protein synthesis. Low food intake of these phenols causes adverse effects in animals similar to protein deficiency (Harper, 1973). The phenol, 3,4-dihyroxyphenylalanine or dopa is an important intermediate in both the synthesis of epinephrine and melanin, in man. It is a derivative of tyrosine and has been used as an antitumour drug for the treatment of melanomas (Wick, 1977; Parsons and Morrison, 1982). Rare autosomal recessive genetic deficiencies in the metabolism of these phenolics may lead to conditions known as phenylketonuria, tyrosinemia, alkaptonuria, and albinism. Interestingly, tyrosinemia which is believed to result from a deficiency in the enzyme, p-hydroxyphenylpyruvate hydroxylase, often leads to hepatic carcinomas in patients at a very young age (Weinberg, et a l . , 1976). Vitamins K and E are essential components of man's diet . Vitamin K is broadly distributed in plants and is required in the diet of most higher animals. It is believed to play a role in prothrombin synthesis. Vitamin E consists of a group of closely related vitamers present in vegetable o i l s , known as tocopherols. Nutrit ional deficiency in this vitamin leads to s t e r i l t y , muscular weakness, and atrophy in rodents (Lehninger, 1970). Strong evidence suggests that i t ' s "antioxidant" properties prevent the destructive non-enzymatic attack of molecular oxygen on the double bonds of polyunsaturated fatty acid components of tissue l i p i d s . It also appears to prevent oxidation of other components of the diet . This w i l l be discussed in some detai l in the Discussion part of this thesis. 3 Although phenols are found i n almost a l l " p l a n t s , there i s great d i v e r s i t y i n the type and content f o r each species. However, the foods a c t u a l l y consumed by higher animals appear to be low i n both v a r i e t y and content of the v a r i o u s forms of p h e n o l i c s . This i s most l i k e l y due t o t a s t e s e l e c t i o n i n animals. Given a c h o i c e , animals choose foods w i t h a low p h e n o l i c content from a l i g n i n (toughness) or t a n n i n (astringency) viewpoint, but perhaps a r e l a t i v e l y high anthcyanin content (colour ripeness) (Bate-Smith, 1972). . ' C l a s s i f i c a t i o n of P h e n o l i c s : P h e n o l i c s are compounds of the general formula, ArOH, where Ar i s a phenyl and OH, a hydroxy1 group. Often these compounds are l i q u i d s or low m e l t i n g s o l i d s and because of hydrogen-bonding, have r e l a t i v e l y high b o i l i n g p o i n t s . T y p i c a l l y they e x h i b i t a c i d i t y w i t h a high r e a c t i v i t y of t h e i r r i n g s t r u c t u r e toward e l e c t r o p h i l i c s u b s t i t u t i o n . Most are only s p a r i n g l y s o l u b l e i n water (Morrison and Boyd, 1973). For s i m p l i c i t y , these compounds have been d i v i d e d i n t o the f o l l o w i n g c l a s s i f i c a t i o n : a) simple phenols, b) p h e n o l i c a c i d s ; i ) benzoic a c i d s and i i ) cinnamic a c i d s , c) f l a v o n o i d s , d) t a n n i n s , and e) l i g n i n s . A. Simple Phenols: Simple phenols may be d e f i n e d as molecules c o n t a i n i n g only the benzenoid r i n g s t r u c t u r e w i t h an adjacent h y d r o x y l group(s). O H 4 Appendix 1 l i s t s the molecular s t r u c t u r e s of some of the more commonly found simple phenols. Because of t h e i r extreme t o x i c i t y , they do not tend to be found i n t h e i r parent form i n p l a n t s . They do, however, e x i s t as breakdown products of l a r g e r , more h i g h l y s t r u c t u r e d phenols and nonphenols. P y r o l y s i s of c a t e c h i n (from gum catechu) and g a l l a t e s were the h i s t o r i c a l sources of c a t e c h o l , a simple phenol of high r e a c t i v i t y ( S i n g l e t o n , 1981). Simple phenols can a l s o be generated by p y r o l y s i s of carbohydrates (Higman e t a l . , 1970). A l l phenols e x h i b i t some degree of a n t i - m i c r o b i a l a c t i v i t y ( F r i e n d , 1981). B. P h e n o l i c A c i d s : i ) Benzoic A c i d s : These compounds possess the same b a s i c p h e n o l i c s t r u c t u r e ; the benzenoid r i n g w i t h a hydroxy1 group, but w i t h the a d d i t i o n of a c a r b o x y l i c a c i d group immediately adjacent to the r i n g s t r u c t u r e . The molecular s t r u c t u r e s and names of commonly found benzoic a c i d s are shown i n Appendix 2. Benzoic a c i d s are w i d e l y d i s t r i b u t e d i n p l a n t s and can be found i n both angiosperms and gymnosperms (Ribe"reau-Gayon, 1972) . The most important mechanism of formation of benzoic a c i d s i n p l a n t s i s the s i d e chain degradation of cinnamic a c i d s (Gross, 1981). C O O H 5 p-Hydroxybenzoic, v a n i l l i c and s y r i n g i c a c i d s are c o n s t i t u e n t s of l i g n i n , from which they are l i b e r a t e d by a l k a l i n e h y d r o l y s i s (Ribe*reau-Gayon, 1972) . Protocatechuic and g a l l i c a c i d are b e l i e v e d t o have a d i f f e r e n t f u n c t i o n . The former i s widely d i s t r i b u t e d while the' l a t t e r i s r a r e r and o f t e n found as i t s dimer, e l l a g i c a c i d . G a l l i c and e l l a g i c a c i d s are important components of many tannins. Bound forms of g a l l i c a c i d , notably e p i c a t e c h i n g a l l a t e or g a l l o y l glucoses and t a n n i c a c i d s are a s s o c i a t e d w i t h and are the main source of g a l l i c a c i d i n foods ( S i n g l e t o n , 1981). The t o x i c i t y l e v e l s f o r benzoic a c i d s are r e l a t i v e l y low i n animal s t u d i e s ( S i n g l e t o n , 1981). They do possess, however, considerable a n t i - m i c r o b i a l a c t i v i t y ( F r iend, 1981). i i ) Cinnamic A c i d s : Cinnamic a c i d s are s i m i l a r i n molecular s t r u c t u r e t o benzoic a c i d s , but possess an ethylene group between the benzene r i n g and the c a r b o x y l i c a c i d group. T h e i r s t r u c t u r e s are i l l u s t r a t e d i n Appendix 2. L i k e benzoic a c i d s , these compounds are widely d i s t r i b u t e d i n nature. The four cinnamic a c i d s most commonly known and d i s t r i b u t e d i n p l a n t s are: p-coumaric, c a f f e i c , f e r u l i c , and s i n a p i c a c i d s . At l e a s t one of these i s b e l i e v e d to be found i n a l l p l a n t s . According to Bate-Smith 6 O H C H C H C O O H (1959) , p-coumaric a c i d i s the most commonly "found p h e n o l i c of a l l p h e n o l i c c o n s t i t u e n t s (Ribereau-Gayon, 1972). Since cinnamic a c i d s possess a double bond, they can e x i s t i n two isomeric forms, c i s and trans-cinnamic a c i d . trans-cinnamic a c i d c i s - c i n n a m i c a c i d The n a t u r a l l y o c c u r r i n g cinnamic a c i d s are the trans isomers. However, these isomers can be i n t e r c o n v e r t e d by the a c t i o n of l i g h t (Ribe"reau-Gayon, 1972). A g r i c u l t u r a l l y important foods c o n t a i n high l e v e l s of cinnamic a c i d d e r i v a t i v e s . These i n c l u d e c a f f e i c , p-coumaric, f e r u l i c , and s i n a p i c a c i d s (or 3,4-dihydroxy-, 4-hydroxy-, 3-methoxy-4-hydroxy-, and 3,5-dimethoxy-4-hydroxy-cinnamic a c i d s , r e s p e c t i v e l y ) . Often these cinnamates occur as e s t e r s of q u i n i c a c i d (chlorogenic a c i d and analogues) or other nonphenolic hydoxyl compounds such as glucose, s h i k i m i c a c i d and t a r t a r i c a c i d (reviewed by S i n g l e t o n , 1981). The common p h e n o l i c cinnamic a c i d s do not occur f r e e l y i n p l a n t s but are a c e t y l a t e d to q u i n i c and other h y d r o x y l i c substances. In animals, q u i n i c and s h i k i m i c a c i d d e r i v a t i v e s are converted to s a t u r a t e d cyclohexane c a r b o x y l i c d e r i v a t i v e s by i n t e s t i n a l microorganisms. They are then metabolized by the l i v e r t o the benzoic a c i d conjugate, 7 h i p p u r i c a c i d (Brewster et a l . , 1977). This i l l u s t r a t e s the f a c i l i t y animals have f o r d e t o x i f y i n g some of the more predominant phe n o l i c compounds. C. Flavonoids: The chemical s t r u c t u r e s of f l a v o n o i d s i s based on a C^ ,. s k e l e t o n w i t h a chromane r i n g b e a r i n g a second aromatic r i n g B i n p o s i t i o n 2,3, or 4. Subgroups of favonoids are c l a s s i f i e d according to the s u b s t i t u t i o n p a t t e r n s of r i n g C. Both the o x i d a t i o n s t a t e of the h e t e r o c y c l i c r i n g and the p o s i t i o n of r i n g B are important i n the c l a s s i f i c a t i o n (Hahlbrock, 1981). Examples of the s i x major subgroups (chalcones and the isomeric f l a v o n e s , f l a v o n e s , f l a v o n o l s , anthocyanins, and i s o f l a v o n o i d s ) are i l l u s t r a t e d i n Appendix 3. Seve r a l reviews e x i s t on the s t r u c t u r e , occurrence, and methods of i d e n t i f i c a t i o n of f l a v o n o i d s (Harborne e t a l . , 1975; Ribereau-Gayon, 1972; Mabry e t a l . , 1970). Common p l a n t foods c o n t a i n from t r a c e s to s e v e r a l grams of f l a v o n o i d s per ki l o g r a m f r e s h weight. A few hundred m i l l i g r a m s per kil o g r a m i s common f o r many vegetables and f r u i t s ( S i n g l e t o n , 1981). The human d i e t a r y i n t a k e has been estimated to be as h i g h as 1 gram per day (Kuhnau, 1976). Flavonoids found i n a g r i c u l t u r a l l y important foods i n c l u d e the common n a t u r a l anthocyanins: c y a n i d i n and d e l p h i n i d i n , the fl a v o n e s : apigenin and l u t e o l i n , the f l a v o n o l s : kaempferol and q u e r c e t i n , the f l a v a n - 3 - o l s : (+)-catechin and (-)-epicatechin, the flavanone: n a r i n g e n i n , and the favanonol: t a x i f o l i n (reviewed by S i n g l e t o n , 1981). Flavonoids are r e s p o n s i b l e f o r the co l o u r and f l a v o u r i n food. They a l s o c o n s t i t u t e the pigmentation i n f l o w e r s . Most f l a v a n o i d s show low t o x i c i t y , however, a number have been shown to induce mutagenicity. The most e x t e n s i v e l y r e p o r t e d , q u e r c e t i n , i s a component of the car c i n o g e n i c braken f e r n (Pamukcu, A.M., '1980) . D. Tannins: The tannins are a d i v e r s e and chemic a l l y d i f f i c u l t group t o c l a s s i f y . T h e i r two main c h a r a c t e r i s t i c s are a p o l y p h e n o l i c chemical s t r u c t u r e , and t h e i r a b i l i t y to p r e c i p i t a t e p r o t e i n from aqueous media. Much of the d i f f i c u l t y i n t h e i r c l a s s i f i c a t i o n has a r i s e n from the f a c t t h a t e a r l y biochemists confused t h e i r a b i l i t y to produce l e a t h e r from h i d e , the tanning process, w i t h t h e i r p o l y p h e n o l i c nature. In t h i s way the accepted meaning of the word, t a n n i n , was enlarged i n the general b o t a n i c a l and biochemical l i t e r a t u r e to i n c l u d e a range of compounds which are polyphenols, but not tannins (Haslam, 1979). The most widely accepted d i v i s i o n i s t h a t f i r s t proposed by Freudenberg i n 1920. I t separates the tannins i n t o two c l a s s e s , the hydrolysable and the nonhydrolysable or condensed tannins. i ) Hydrolysable Tannins: The h y d r o l y s a b l e t a n n i n s , as t h e i r name i n f e r s , are complex phenols which can be degraded under h y d r o l y t i c c o n d i t i o n s ( a c i d , a l k a l i , or h y d r o l y t i c enzymes). S u b d i v i s i o n i s u s u a l l y made on the b a s i s of the ph e n o l i c a c i d ( s ) l i b e r a t e d by h y d r o l y s i s . Those y i e l d i n g g a l l i c a c i d as the only p h e n o l i c component are def i n e d as g a l l o t a n n i n s and those 9 l i b e r a t i n g besides g a l l i c a c i d , i t s d e r i v a t i v e ' e l l a g i c a c i d , are named e l l a g i t a n n i n s . o II H O H O i O H O g a l l i c a c i d e l l a g i c a c i d G a l l o t a n n i n s are e s t e r s of sugars, such as glucose, and g a l l i c a c i d or m - d i g a l l i c a c i d . They are not widely d i s t r i b u t e d i n p l a n t s (Haslam, 1981). E l l a g i t a n n i n s , on the other hand, do not n e c e s s a r i l y combine w i t h glucose i n the o r i g i n a l t a n n i n . These tannins are more widely d i s t r i b u t e d (Haslam, 1981). i i ) Condensed Tannins: These tannins are the most commonly found and are of the g r e a t e s t commercial s i g n i f i c a n c e (Haslam, 1981). They are widespread i n f r u i t s and c e r t a i n g r a i n s as w e l l as i n such beverages as c i d e r , cocoa, t e a , and red wine (Haslam, 1975). R e l a t i v e l y l i t t l e i s known, however, about t h e i r s t r u c t u r e and molecular formation. Freudenberg (1920) f i r s t put f o r t h h i s " c a t e c h i n hypothesis" i n which (+)-catechin formed the sole b a s i s of tannin chemistry. Wide ranging s t u d i e s w i t h f l a v a n - 3 - o l s and f l a v a n - 3 , 4 - d i o l s as models has l e d to proposals f o r t h e i r s e l f - c o n d e n s a t i o n to the complex polymers known as condensed tannins (Haslam, 1966). 10 O H O H O H f l a v a n - 3 - o l s (catechins) f l a v a n - 3 , 4 , d i o l s (leucoanthocyanidins) For a review of t h i s h y p o t h e s i s , see Haslam, 1981. The d i s t r i b u t i o n i n nature of condensed t a n n i n s , based on leucoanthocyanidins has been examined by Bate-Smith and Metcalfe (1957) and by Bate-Smith and Ribe'reau-Gayon (1958) . These compounds were found t o be so prominent, t h a t t h e i r property of g i v i n g anthocyanidins upon hea t i n g w i t h a c i d c o u l d be used as a d i a g n o s t i c t o o l f o r t h e i r presence (Swain, 1975). Condensed tannins have a molecular weight of between 500 and 3000. Since the f l a v a n s , diagrammatically represented above, have molecular weights i n the order of 250-300, the condensed form would c o n t a i n 2 to 10 monomers and can thus be considered as oligomers (Ribe'reau-Gayon, 1972). P o l y m e r i z a t i o n p l a y s a r o l e i n the p r o p e r t i e s of these compounds. Whether dimers, t r i m e r s or tetramers are present during formation, has been found to a f f e c t the shape of the subsequent molecule, and thus i t s p r o p e r t i e s of s t a b i l i t y , d i s t r i b u t i o n , and r e a c t i o n w i t h p r o t e i n (Ribe"reau-ayon, 1972) . Tannins have played an important r o l e i n f o l k medicines. Because of t h e i r potent a n t i m i c r o b i a l a c t i v i t y , they have been used as a n t i b i o t i c s (Chan et a l . , 1978), as a burn treatment, and as an enema ( S i n g l e t o n , 11 1981). T h e i r property of b i n d i n g p r o t e i n enhanced coagulation o f a burned surface and produced l o c a l inflammation, a property which enabled them to be used as a form of enema. However, at the time of the second world war, they were a l s o found to induce h e p a t o t o x i c i t y and have si n c e lapsed i n t o d i s u s e . This h e p a t o t o x i c i t y i s most l i k e l y r e l a t e d t o f i n d i n g s of c a r c i n o g e n i c i t y by Kdrpassy, 1961. K i r b y , 1960, has suggested t h a t condensed tannins are bound to body p r o t e i n s , where, as hydrolysable t a n n i n or fragments, they may be t r a n s l o c a t e d to the l i v e r . E p i d e m i o l o g i c a l s t u d i e s have attempted to evaluate the r o l e of tannins i n terms of human cancer. Some r e p o r t s have c o r r e l a t e d the frequency of mouth and esophageal tumours w i t h the consumption of t a n n i n - r i c h m a t e r i a l (Morton, 1970; 1972; 1973; Raghava and Baruah, 1958). These c o r r e l a t i o n s have been examined i n the cases of b e t e l nut chewing i n p a r t s of A s i a and the consumption of sorghums i n p a r t s of A f r i c a , and h e r b a l teas on other areas ( S i n g l e t o n , 1981). E• L i g n i n s : L i g n i n i s found as an i n t e g r a l c e l l w a l l c o n s t i t u e n t of a l l v a s c u l a r p l a n t s . I t i s comprised of a heterogeneous group of l a r g e , i n s o l u b l e , t h r e e - d i m e n s i o n a l l y l i n k e d polymers. U n l i k e p o l y s a c c h a r i d e s such as c e l l u l o s e i t l a c k s an ordered s t r u c t u r e . I t i s b e l i e v e d t h a t i t i s formed by enzymatic dehydrogenation of cinnamyl a l c o h o l s (example, 4-coumaryl a l c o h o l ) , c o n i f e r y l a l c o h o l , and s i n a p y l a l c o h o l (Grisebach, 1981) . 12 C H p H -O H R£=R2=H; 4-coumaryl a l c o h o l R1=OCH3, R2=H; c o n i f e r y l a l c o h o l R =R =OCH ; s i n a p y l a l c o h o l L i g n i n , or l i g n i n f i b r e s have never been shown t o be t o x i c t o man (Si n g l e t o n , 1981) They are a component of d i e t a r y f i b r e and as such add bulk to the d i e t of higher animals. They a l s o appear to absorb b i l e s t e r o i d and d i e t a r y c h o l e s t e r o l , as w e l l as bi n d n i t r i t e therebye i n h i b i t i n g nitrosamine formation i n the gut ( I n g l e t t and Falkehag, 1979). L i g n i n ' s r o l e i n d i e t may t h e r e f o r e be fa v o r a b l e , or at l e a s t i n e r t ( S i n g l e t o n , 1981). T e s t i n g of P h e n o l i c s : P l a n t p h e n o l i c s to be screened i n a short term t e s t f o r c l a s t o g e n i c a c t i v i t y were those found to be present i n foods from a North American d i e t . Of p a r t i c u l a r i n t e r e s t were those present i n p h e n o l i c - r i c h beverages which have been l i n k e d i n e p i d e m i o l o g i c a l s t u d i e s t o human cancers. Coffee has been i m p l i c a t e d as an e t i o l o g i c a l l y important f a c t o r i n the development of tumours of the u r i n a r y bladder (Cole and MacMahon, 1971; Simon et a l . , 1975; Howe et a l . , 1980) pancreas (MacMahon et a l . , 1981) and ovary (Trichopoulos et a l . , 1981). The use of h e r b a l teas has been i m p l i c a t e d w i t h the development of 13 nasopharyngeal cancers in China (Hirayama and "ltd, 1981) and Curacao (Hecker, 1981). "Chagayu", a r ice gruel boiled with tea leaves, has been associated with an elevated r i sk of esophageal tumours (Segi, 1975, Hirayama, 1979). These phenolic-rich beverages are particularly high in content of chlorogenic acid and i t s breakdown product, caffeic acid (the average cup of coffee contains 260 mg or about 10 mg/ml) (Challis and Bartlett , 1975) and polyphenols (teas may contain up to 30% per dry weight) (Kaiser, 1967). These compounds were also readily available and showed considerable ease of handling. The CHO C e l l Chromosome Aberration Test: The introduction of short-term tests for the screening of potentially hazardous substances has greatly aided in the identif icat ion and c lass i f icat ion of carcinogens. They accomplish this by circum-venting the enormous cost and time required for l ive animal testing. At present, the most widely used assay is the Ames' bacterial mutation system. In surveys conducted to correlate the results of the Ames test with those of in vivo tests, the Ames test was found to correctly identify about 90% of the compounds tested (McCann et a l . , 1975; Coombs et a l . , 1976; Anderson and Styles, 1978). It should be pointed out, however, that these tests are not effective for screening a l l carcinogens. One such group of compounds are the oxidative mutagens, substances capable of giving rise to activated oxygen species. Testing in this lab demonstrated that the CHO c e l l chromosome aberration test was capable of detecting these substances. Recently, the Ames group has developed a new s tra in , TA102, which i s sensitive to these compounds; however i t was unavailable at the time of testing (Levin et a l , 1982). 14 The CHO c e l l chromosome test assays Tor the induction of chromosomal damage. Data on chromosome breakage can therefore be compared with the bacterial mutation assays. In a paper by Ishidate and Odashima (1977) 134 chemicals were assayed for their cytogenetic effects. .Thirty four of the test chemicals had been previously demonstrated to be carcinogenic. The chromosome aberration test correctly identif ied 25 of the 34 (73.5%) and incorrectly identif ied 9 (26.5%). These results were comparable with the Ames system which correctly identif ied 28 (82.4%) and incorrectly identified 6 (17.6%). Aside from being sensitive to oxidative clastogens, the CHO c e l l test system offers many other advantages. Almost a l l c e l l l ines derived from Chinese hamster tissues have a stable karyotype with a low diploid number (2n = 22, Au and Hsu, 1982). Although this c e l l l ine tends to become aneuploid with time, this does not pose a serious problem, since clastogen effects do not require analyses of chromosome numbers. C e l l cycle times for this particular l ine is about 14 hours, a time l imit which is easy to incorporate into a laboratory schedule. Scoring for this test was done by recording the number of breaks, gaps and translocations per c e l l in mitosis (see Figure 1). Application of the test chemical was for 3 hours with a subsequent 16 hour incubation period. We would, therefore, anticipate only ce l l s damaged orig inal ly in late S phase to be scored in mitosis. One would therefore only expect chromatid as opposed to chromosome aberrations. While longer incubation times with test chemicals would i l lus tra te i f damage could be induced ear l ier in the c e l l cycle, i t was not in the realm of this study to examine stage-specific agents. The CHO c e l l test system employed was one for the rapid identif icat ion of oxidative clastogens. 15 Figure 1: Or c e i n - s t a i n e d metaphase CHO c e l l s which have been exposed to a c l a s t o g e n i c agent f o r 3 hours. The c e l l s were then washed, c u l t u r e d f o r an a d d i t i o n a l 16 hours, exposed to c o l c h i c i n e f o r 4 hours and s t a i n e d and mounted. "A" r e f e r s t o c o n t r o l chromosomes, and "B" are chromosomes which have been t r e a t e d w i t h t e s t chemicals. Note chromatid breaks and t r a n s l o c a t i o n s (marked w i t h arrows). 16 17 Identification of Hydrogen Peroxide; Several techniques have been employed for the measurement of ^2®2 in solution. Cytochrome c peroxidase in the presence of H2O2 forms a stable enzyme substrate complex with ferrocytochrome c. Inhibition of the reaction is accomplished by the addition of cyanide or low azide concentrations. This technique is extremely sensitive (capable of detecting formation at a rate of 1 uM/min.) although s l ight ly complex (Boveris et a l . , 1972; Yonetani, 1965). Because of the large quantities of l^C^ produced under our experimental conditions, this technique was deemed unsuitable. Another technique measures H2O2 production by the detection of generated in the presence of catalase (Jensen, 1966). This is accomplished with an oxygen electrode, a piece of equipment not available at the time of this study. Production of 2^^ 2 W a S therefore measured in a re lat ively inexpensive fashion as described below. In this study, the assay employed for the identif icat ion of H2O2 was one modified from Wang and Nixon, 1978. This assay contains potassium iodide (KI) and trace amounts of ammonium molybdate at pH 5.0. Under these conditions, there is v i r tua l ly no absorbance at 360 nm in an absorbance spectrophotometer. Peroxides, catalyzed by the presence of ammonium molybdate react with the iodide to generate I^ which absorbs l ight at a maximum of 360 nm. Controls for this reaction were obtained by preparing identical samples in the presence of catalase, an enzyme which catalyzes the destruction of 2^^ 2 t 0 ^2^ and O2. The production of I^ could then be obtained using the net absorbance readings at 360 nm. Conversion of this value to molar quantities of 2^^ 2 W a s obtained by comparing absorbance values obtained from test chemicals with a standard curve generated in the presence of commercially available ^2^2' ^ i s system is capable of 18 measuring quantities of HO in the 0 - 100 uM range. Identification of Superoxide: Because of the rapidity of spontaneous dismutation of ^2'' steady state concentrations of this radica l , whether enzymatically or chemically produced, tend to be relatively low. Chemical methods for the detection of O2* are integrative and allow great sensit ivity for i t s detection. In these methods, 0^ * is trapped with a chosen scavenger. The reaction is then followed by changes in absorbance as the scavenger alters i t s electronic configuration. The scavenger is used at concentrations which compete with the dismutation reaction so that v i r tua l ly a l l 0^ * present is trapped by the scavenger. Several forms of scavengers have been employed for the detection of O2'• The O2" dependent oxidation of epinephrine to adrenochrome may be followed spectrophotometrically (McCord and Fridovich, 1969). However, the speci f ic i ty of this reaction is re lat ively poor and the autooxidation of epinephrine must be controlled by lowering the pH (Bors et a l , 1978). This prohibits any study of rates of production of O2" at different pH values. The rate of reduction of nitrobluetetrazolium has also been employed as an indication of O2" production but this method also appears to lack speci f ic i ty (Beauchamp and Fridovich, 1972) . The method of choice, then i s the measurement of reduction of ferricytochrome c. The rapidity of this reaction makes cytochrome c an excellent quantitative trap (Forman and Fridovich, 1973). Acetylation of the cytochrome molecule has been employed for greater specif ic i ty in mitochondrial measurements of ^2' production (Azzi et a l . , 1975). However, this was not required in this present study, because the test conditions used lacked any electron transport chain enzymes which might 19 reduce the speci f ic i ty of the reaction. In this reaction, superoxide anion radicals reduce native ferricytochrome c(cyt 3+) by univalent electron transfer: 3+ 2+ 0 2 + cyt • cyt + 0 2 (from Kuthan and U l l r i c h , 1982). The reduced cytochrome molecule has a characteristic absorption maximum at 550 nm. Since the molar extinction coefficient for the reduced form is well known, and one molecule of ferricytochrome C + 2 is produced for each molecule of superoxide present, the concentration of superoxide anion radicals can be estimated. Generally, the rate of production is expressed as the rate of conversion at half maximum absorbance, or KQ (Finkelstein et a l . , 1981) (see Results). Measurement of Phenolic Free Radicals: The electron spin resonance, or ESR measurement of free radicals was f i r s t performed by Zavoisky in 1945 (Zavoisky, 1945). Since then, greater sensi t iv i ty has been obtained through better instrumentation and techniques of handling samples. ESR is now considered the single most important experimental approach to research on free radicals (Borg, 1976). The phenomenon of ESR occurs because a l l electrons have identical values of mass, charge, in tr ins ic angular momenta and magnetic moments which permit them to interact with their atomic or molecular surroundings in ways characteristic of the chemical structure of the molecule to be studied. The c r i t i c a l property which underlies these measurements is the quantized value of the electron's magnetic moment, a value derived from i ts spin. 20 The electron to be measured is a moving" charge and therefore generates a magnetic f i e l d , the axis of which has an associated magnetic dipole moment. Since quantum restrict ions require a l l electrons to be the same, relative to any axis of reference, only two orientations of spin are allowed for each atom. These two orientations are energetically equal in the absence of an external magnetic f i e l d . However, in an applied magnetic f i e ld they act to align in either a para l l e l or ant iparal le l fashion. In the para l le l state, the electron is energetically more stable and is thus at lower energy than the ant iparal le l state. The equation which describes the energy difference between states is given below: AE = gBH where E = energy g = g-factor (a proportionality constant) B = Bohr magneton (a constant) H = magnetic f i e ld In an idealized "free electron" sample, one would expect the value of AE to be constant from one electron to another. However, in the presence of other electrons and their magnetic states, the value of AE becomes characteristic of the molecule. Electron spin resonance spectroscopy takes advantage of the fact that only paramagnetic molecules - or odd electron sites - can be made to interact with appropriate external f ie lds . Thus, this method allows one to examine the molecular surroundings of the free radical in a bulk sample matrix. ESR spectra provide information in a number of ways. F ir s t of a l l , the applied magnetic f i e ld at which resonance occurs is largely determined by the effective spectroscopic sp l i t t ing factor, the 21 g-factor. This value is a constant of the free~radical studied and can be used for comparison of the experimental conditions used by other groups. A l l g-factors measured in this study were obtained using the marker diphenylpicrylhydrazyl (DPPH) which has a known g value of 2.0036 ± 0.0002 (Ki t t e l , 1971). Calculations of g-factor were based on the equation: gx = gDPPH, 1 + AH corr HD where gDPPH = 2.0036 ± 0.0002 AHcorr = corrected f i e ld (G) = f i e ld of DPPH (G) The number of spectral l ines produced in an ESR spectrum can also provide valuable information about the radica l . These reflect interactions of unpaired electrons with localized magnetic f ields which are not a result of the applied external f i e l d , and are referred to as the nuclear hyperfine interactions. This sp l i t t ing of resonance lines is dependent on atoms which possess inherent nuclear spin angular momenta such as ^ ^ D , 1 I + N , and 1 3 C . Since nuclear spin moments are quantized properties, the number of spl i t t ings is discrete and may be defined by the equation: N R f s = (21 + 1) where N H f g = number of hyperfine spl i t t ings I = nuclear spin quantum number 22 In the phenolic samples measured, only *H contributed to the hyperfine sp l i t t ing phenomena. The separation of these spectral lines is a reflection of the strength of the nuclear hyperfine interaction. Values for Njj^ g for the phenolics measured in this study were available in the current l i terature and i t was possible to correlate the values obtained with those published. F ina l ly , the intensity of the signal provides information about the number of free radicals present. Signals obtained by autooxidation at alkaline pH-values were observed to be s ignif icantly stronger than those generated enzymatically. However, both appeared to exhibit similar hyperfine patterns and g-values. Using the electron spin resonance technique i t was possible to delineate some of the characteristics of the phenolic free radical molecules. It is the contention of this thesis that the oxidative products of plant phenolics, namely, hydrogen peroxide, superoxide and phenolic free radicals , contributed to the clastogenic act iv i ty of phenolic molecules in a CHO c e l l chromosome aberration test. Since these oxidative products have been shown to cause chromosomal damage, i t i s hypothesized that phenolic molecules under oxidative conditions may act as promotors of carcinogenesis. 23 MATERIALS AND METHODS  Ce l l Cultures: CHO cel l s were grown in MEM (Eagle's Minimal Essential Medium, Grand Island Biological Co.) supplemented with 10% fetal calf serum, antibiotics (streptomycin sulphate, 29.6 ug/ml; p e n i c i l l i n , 125 ug/ml; kanamycin, 100 ug/ml; fungizone, 2.5 ug/ml;) and sodium bicarbonate (1 mg/ml). The stock cultures were maintained in 240 ml plast ic culture flasks (Falcon) at 37 C in a water-saturated CO2 incubator. Chemicals: Simple phenols (catechol, resorcinol , pyrogallol) , phenolic acids (p-hydroxybenzoic acid, protocatechuic acid, g a l l i c acid, p-coumaric acid, caffeic acid, ferul ic acid, chlorogenic acid), flavonoids (quercetin, (+)- catechin and (-)-epicatechin) and tannic acid were purchased from the Sigma Chemical Co. (St. Louis, MO.). The purity of these compounds was ascertained by reversed-phase high pressure l iquid chromatography analyses at the wavelength maximum for each of these compounds, No impurities could be detected at this level of analyses. Horse radish peroxidase (Type II , from horse radish, 200 purpurogallin units per mg so l id ) , catalase (purified powder from bovine l i v e r , 11000 Sigma units per mg so l id) , superoxide dismu'tase (Type I, from bovine blood, 2900 Sigma units per mg protein), and tyrosinase (Grade III from mushrooms, 2000 Sigma units per mg solid) were also purchased from the Sigma Chemical Co . , as were Aflatoxin B l , ferricytochrome c (Type VI, from horse heart), xanthine, xanthine oxidase (Grade III from buttermilk) potassium iodide, and ammonium molybdate. A l l other chemicals were reagent or equivalent grade. 24 Cel l Cultures and Assay for Chromosome Aberrations: The method employed was that previously described by Stich et a l . , 1979. CHO c e l l cultures were grown as 40-60% confluent cultures on 22 mm square coverslips in 35 mm diameter tissue culture dishes. These were exposed to chemicals for a period of 3 hours at 37° C. Chemicals were routinely dissolved in 2.5% MEM (MEM supplemented with 2.5% fetal calf serum) adjusted to pH 7.4, and added to culture dishes in 1 ml aliquots. Tests involving an S9 activating mixture, used 0.5 ml aliquots added to each petr i dish prior to the addition of 0.5 ml of a double strength test chemical. Transition metals and enzymes were added to ser ia l ly diluted stock solutions in 10 ul aliquots just prior to incubation. After incubation, test solutions were removed by gentle suction and cultures were washed twice with warmed fresh MEM, then cultured a further 16 hours in fresh medium supplemented with 10% feta l calf serum. Chromosome aberrations were estimated after a 4 hour incubation period with 0.1 ml of colchicine (0.1% in 2.5% MEM) added to the existing 1 ml volume of 10% MEM. Cel ls on the 22 mm coverslips were treated with a 1% sodium citrate solution for 20 minutes, then fixed in a 3:1 ethanol/glacial acetic acid solution for 20 minutes. Air-dried sl ides were stained with a 2% solution of orcein in 50% acetic acid/water, dehydrated, and mounted in permount. For each sample, a minimum of 100 metaphase plates were analyzed, scoring a c e l l as positive i f i t contained at least one chromatid break or exchange. Exchanges scored included chromatid and chromosome exchanges, mono- and mult i -radials , and rings (see Figure 1). S ta t i s t i ca l variance in the response of the CHO c e l l chromosome aberration test is suff ic iently large to make conventional testing for standard deviation impossible. To provide some idea of the reproducibil ity of the test, sample compounds were tested on the same 25 day and again on alternate days in double blind experiments. These results are tabulated in Table 1, and provide the background spontaneous breakage rate of a c e l l l ine scored under the experimental conditions l i s ted above. Controls for CHO Ce l l Conditions: The act iv i ty of the S9 activation mixture was assayed with the -2 following controls. A stock solution of 2x10 M Aflatoxin Bl (Sigma' Chemical Co . , St. Louis, M0.)> was ser ia l ly diluted to concentrations of -4 -5 -5 2x10 M, 2x10 M, and 1x10 M in 2.5% MEM. 0.5 ml of these solutions were then added with 0.5 ml of the S9 mixture or MEM to a -4 -5 -6 f ina l concentration of 1x10 M, 1x10 M, and 5x10 M aflatoxin B l . The S9 was deemed satisfactory i f at least 80% of the metaphase plates had at least one chromosome break at a concentration of 5x10 ^ aflatoxin B l . Aflatoxin Bl at the same concentration, but without S9 present should not elevate the frequency of chromosome aberrations over that found in non-treated control cultures. The enzymes horseradish peroxidase, catalase, superoxide dismutase, and tyrosinase were also added separately at the same concentrations as were used with test chemicals, to check for any increase in the spontaneous frequency of chromosome breaks. A similar procedure was used for transit ion metal controls of copper sulphate and manganese chloride. Preparation of S9: The S9 mixture was prepared as described by Ames et a l . , 1975. Microsomal preparations were obtained from the l ivers of male Swiss rats that had been pretreated with Aroclor 1254. The S9 mixture consisted of l iver supernatant (0.3 ml per mix), 0.4 M MgCl 2 , 1.65 M KC1, glucose-6-phosphate (1.3 mg/ml mix), NADP (2.55 mg/ml mix) and 0.2 M 26 phosphate-buffered saline. This solution was freshly prepared and 0.5 ml was added to each petri dish prior to the addition of 0.5 ml of double strength test chemical. Preparation of Metal Solutions: 0.1 M solutions of cupric sulphate, and manganese chloride were prepared with glass d i s t i l l e d water. These stock solutions were then diluted 10-fold in a 0.5 M stock solution of glycine. 10 ul of the resulting solution was added to each 1 ml aliquot of the test compound -4 to give a f i n a l concentration of lx 10 M transition metal content. High Pressure Liquid Chromatography: High Pressure Liquid Chromatography (HPLC) chromatograms were obtained using a Perkin Elmer Series 2 gradient pump, under the direction of a Perkin Elmer Sigma 10 data system. Absorption was monitored at 260 nm with a Perkin Elmer LC-55 absorption spectrophotometer. Chromatography was carried out at a flow rate of 2 ml/mn on a Vydac 201 TP 5 micron reversed phase column, 4.6x250 mm. A linear gradient starting at 2% methanol, 98% glass d i s t i l l e d water and r is ing by 2% methanol per minute was employed. A l l solvents contained 0.1% phosphoric acid to suppress ionization of phenolic acids and permit reversed phase chromatography. This level of phosphoric acid was not found to cause chromosome aberrations in control CHO cultures. Separation of oxidation products for testing on CHO c e l l chromosomes was accomplished using a Waters preparative reversed phase column on a Waters Prep LC/System 500A preparative HPLC system. Samples were eluted at a flow rate of 100 ml/min at an isocrat ic methanol concentration of 20%. Concentrates were obtained by rotary evaporation at 30°C unt i l a l l methanol was removed. The samples so obtained were then run on Vydac 201 TP 5 micron columns a"s described above for identif icat ion and purity purposes. Assay for Hydrogen Peroxide: Hydrogen peroxide (H^O )^ was measured using a modification of the procedure described by Wang and Nixon (1978). Colour reagent was prepared fresh each day, and contained 6% potassium iodide, 0.05 M acetic acid, and 2x10 M ammonium molybdate. Aliquots (0.4 ml) of the sample to be assayed were pipetted into two separate tubes. Catalase (0.1 ug in 10 yl) was added to one sample, and the sample allowed to incubate at room temperature for a minimum of 10 minutes. This period of time was sufficient to destroy H^O^ in concentrations of up to 100 uM. Aliquots (0.4 ml) of colour reagent were then added to each sample, and the samples incubated at room temperature for a further 5 minutes. Absorbance resulting from the molybdate catalyzed release of I^ by was determined at 360 nm in a dual beam Perkin Elmer Lambda 3 spectrophotometer, using the peroxide-free catalase-treated sample as a blank. ^2®2 l e v e l s were estimated from a standard curve prepared using phosphate buffered saline/dextrose samples containing 0-100 micromolar H^O^ (see Figure 2). When necessary, samples were diluted before the assay to bring the 1^ 02 levels to within the range of the standard curve. Samples containing high levels of caffeic acid were also diluted before assay to bring the blank absorbance at 360 nm to within the range of the spectrophotometer. Assay for Superoxide: • The method employed was a modification of that described by McCord and Fridovich, 1969. Superoxide, ^ ' , formation was measured as the rate of reduction of ferricytochrome c (Type VI , from horse heart) 28 Figure 2: Reference curve for the determination of concentration. Known quantities of commercially-available H 2 ° 2 w e r e assayed with the potassium iodide colour reagent and plotted vs. absorbance at 360 nm. The dotted l ine insert is a magnification of values below 6 uM of H 2 ° 2 " T n i s assay was linear between 1 and 80 uM, any measurements above this level had to be diluted back into this range. 29 Absorbance (O.D. units) reflected as an increase in absorbance at 550" rim. Caffeic acid was freshly prepared as a 1x10 M solution in 0.05 M phosphate buffer at a f inal pH of 5.0. One half of this mixture was taken and to i t was added sol id superoxide dismutase (Type I , from bovine blood) to a f ina l concentration of 50 yg/ml. This mixture was allowed to incubate at room temperature for a minimum of one hour. Absorbance spectra were scanned from wavelengths of 400 rim to 700 nm (the v i s ib le region) immediately prior to in i t ia t ion of the assay. This precaution ensured that no oxidation of the caffeic acid solutions had occurred, since oxidation products of caffeic acid tend to absorb maximally in this range. A cytochrome c stock solution was prepared at a concentration of 1.1x10 ^ M in 0.05 M phosphate buffer at a pH of 7.0, wrapped in aluminum f o i l , and stored on ice. Using a Perkin Elmer dual beam Lambda 3 absorption spectrophotometer, 2 cuvettes ( 1 cm light path, 2 ml working volume) were zeroed at 550 nm with 0.9 ml of the cytochrome c mixture in each. To one of the cuvettes 0.1 ml of 0.05 M phosphate buffer pH 7.0 was added. To the other cuvette, 0.1 ml of the caffeic acid solution pH 5.0 was added. The f ina l concentrations were thus -4 -5 1x10 M caffeic acid, and 1x10 M cytochrome c in the sample cuvette and 1x10 M cytochrome c in the reference cuvette. The f ina l pH of the solution was measured to be 6.99. Neither freshly prepared caffeic acid nor superoxide dismutase had any appreciable absorbance at 550 nm. The l i d was closed and the chart recorder was begun immediately. The chart recorder was run at 60 mm/min with a f u l l absorbance range of 0.5 optical density (OD) units . This procedure was repeated for the caffeic acid stock solution containing superoxide dismutase. Subsequent measurements were made in an identical fashion but with cytochrome c stock solutions of pH 8.0 and 31 9.0. The addition of 0.1 ml of the buffered caffeic acid solution was found to decrease the pH of the cytochrome solution to pH 7.98 and 8.97 respectively. Rates of reduction of ferricytochrome c were calculated using the equation of Kuthan and U l l r i c h (1982): AAbs x 1_ = concentration of ferricytochrome = KQ 5 t E c at half maximum where AAbs = change in half maximum absorbance at 550 nm t = time (in minutes) at which half maximum absorbance was reached. e = molar extinction coefficient of ferricytochrome c. = 21 T U M - 1 cm"1 (Van Gelder and Slater, 1962). To test for responsiveness of the ferricytochrome c assay to the production of 0^' and i t s destruction by superoxide dismutase, the following technique was employed (McCord and Fridovich, 1969). A stock solution of 1x10 M ferricytochrome c and 5x10 ^ M xanthine was prepared in 0.05 M phosphate buffer pH 7.8 and stored on ice. 2 ml of the stock solution was pipetted into each of two cuvettes and the absorbance level was zeroed for 550 nm. To the sample cuvette was added sufficient xanthine oxidase to provide a rate of reduction of ferricytochrome c of 0.025 optical density units per minute. (20 ul of a 10 ^ M solution of xanthine oxidase). Addition of superoxide dismutase was made in 10 ul aliquots of a 0.10 yg/ml solution in order to determine the amount required to inhibit this rate by 50% (see Figure 3). 32 Figure 3: Reference curve for the measurement of 0^ " and i t s inhibit ion by superoxide dismutase. Curve "a" was prepared by mixing a stock solution of xanthine and xanthine oxidase with a known concentration of buffered ferricytochrome c and monitored as change in absorbance at 550 nm. This served as a 0^ * generating system. Curve "b" was generated by adding a sufficient quantity of superoxide dismutase to inhibit the conversion of cytochrome c by 50%. This provided an assay for the act iv i ty of the enzyme and confirmed that our ferricytochrome c was active. 33 l.OOi I i r 1 2 3 4 Time (minutes) 34 Electron Spin Resonance: Electron Spin Resonance, ESR, studies were performed on a Varian E-Line Century Series Model E-109 EPR System with a TM^ cavity. The microwave cavity was tuned i n i t i a l l y by setting the Varian E102 Microwave Bridge from standby to tune mode, with a 9.52 GHz frequency, 0.2 mW of power with the reference arm in the "off" position and the phase in the mid range. The f ie ld controller module was set with a f i e ld of 03400 gauss, and a scan range' of 4.0 x 100 gauss. Using the high frequency module, the gain was set at 1.25 x 100, with a function 100 KHz and a modulation of 0.80 x 10 gauss peak to peak, and a time constant of .250 seconds. Phenolics were observed to resonate in the range of 3290-3310 gauss f ie ld setting at a microwave frequency of 9.38 GHz. A l l measurements were on aqueous solutions at room temperature in Varian quartz f lat ce l l s which had been cleaned with concentrated suphuric acid and rinsed with d i s t i l l e d water then acetone and blown dry. The reference arm was locked in by inserting the f lat c e l l and tuning the microwave cavity to maximum efficiency. The phase control was adjusted to maximum, and the bridge was switched to the operate position. The microwave power was then increased to 100 mW and the wand adjusted to bring the detector into the range of 200 KHz. The power was then reduced to 0.2 mW and the reference arm was turned unt i l the "lock" position light came on. The microwave power could then be adjusted to the desired setting without causing the receiver level to increase. As a control for the accuracy of the f i e ld controller module, two standards of known g-value were run under identical conditions. These were di-test-butyl nitroxide in CCl^ and crystal l ine diphenylpicryl-hydrazyl (DPPH). The separation of peaks could then be recorded and the f ie ld sweep tuned so that they were in agreement. 35 For g-value measurements, diphenylpicrylhydrazyl was used as an external standard. The DPPH sample was loaded in a quartz capil lary tube and taped to a f lat c e l l . Concurrent measurements of test chemical and DPPH were run in a dual wave cavity. Correction for sl ight variations in magnetic f i e ld for each side of the cavity was measured by placing samples of DPPH on both sides of the dual cavity and recording the f i e ld separation between peaks. Phenolic samples which were oxidized enzymatically required s ignif icantly higher power to generate signals. For these samples, ESR spectra were run at 20 mW. A l k a l i generated signals were recorded at 1 mW of power. 36 RESULTS The Clastogenic Activi ty of Plant Phenolics; The ab i l i t y of plant phenolics to induce chromosome damage was monitored with a conventional CHO c e l l chromosome short term test. I l lustrat ion of the background spontaneous breakage rate of the c e l l l ine , may be found in Table 1. S ta t i s t i ca l variance in the response of the CHO cel ls to chemicals tested, is suff iciently large to make conventional testing for standard deviation impossible. Table 1 i l lus trates , however, that the response of the cel ls is consistent i f the dose level is considered. The clastogenic act iv i ty (ie. the ab i l i ty to cause chromosome damage) of compounds were therefore compared on the basis of the minimum dose found to i l l i c i t ac t iv i ty . The clastogenic act iv i ty of some of the phenols, phenolic acids, flavonoids, and tannins in a CHO c e l l test system may be found l i s ted in Table 2. This table represents a generalized survey of commonly occurring phenolics, and the dose level at which they were found to cause ac t iv i ty . In the group of simple phenols, the act iv i t ies of catechol and resorcinol contrast each other. At dose levels of 0.063 mg/ml catechol, a compound with two adjacent hydroxyl groups, was found to be strikingly active. This act iv i ty was v i r tua l ly lost with the addition of S9 l iver microsomal preparation. Resorcinol, a phenol with hydroxyl groups in the meta positions, was not active unt i l a much higher dose of 2.0 mg/ml was reached. This act iv i ty was enhanced by the addition of S9. A similar trend was observed within the phenolic acid groups of benzoic and cinnamic acids. p-Hydroxybenzoic acid, a benzoic acid with a single hydroxyl group in the para posit ion, was v i r tua l ly inactive even at doses as high as 25.0 mg/ml. S9 s l ight ly enhanced i t s effect. Gal l ic acid however, with three adjacent hydroxyl groups, was extremely TABLE 1: Reproducibility of the CHO c e l l chromosome aberration test. Plate % Metaphases with Chromosome Aberrations Number day 1 day 2 day 3 1 0.0 0.0 0.0 2 0.0 0.0 0.5 3 0.0 1.0 0.0 4 0.0 0.0 0.0 5 0.0 0.0 0.0 6 0.5 0.0 0.0 7 0.5 0.0 0.0 8 0.0 0.0 0.0 9 0.0 1.0 0.5 10 0.0 0.0 0.0 Control test: CHO cel ls were exposed for 3 hours to 2.5% MEM in the absence of test chemical. The background spontaneous breakage rate of the c e l l l ine on day 1, 2, and 3, may be calculated to be 0.1%, 0.2%, and 0.1%, respectively. Concentration % Metaphases with Chromosome Aberrations (mg/ml) day 1 day 1 day 2 2.0 T T T 1.0 T/MI T T 0.5 65.9 T/MI 68.0 0.25 50.0 65.0 52.0 0.125 7.5 8.0 16.0 0.06 3.2 3.4 5.5 0.03 0.0 0.0 1.0 Control test: CHO cel ls were exposed to the same ser ia l dilutions of caffeic acid concurrently, and on separate days. T represents toxic i ty , and T/MI is used to describe levels of test chemicals which caused toxicity and mitotic inhibit ion in metaphase chromosomes which were scored. 38 TABLE 2: The clastogenic act iv i ty of phenolics in a CHO c e l l test system. 1 % Metaphases Phenolic Compound Concentration Activation with Group (mg/ml) Chromosome . Aberrations Simple Catechol 0.063 -S9 32.0 Phenols +S9 3.0 Resorcinol 2.0 -S9 2.0 +S9 10.0 Phenolic p-Hydroxybenzoic 25.0 -S9 0.0 Acids Acid +S9 8.5 i) Benzoic Gal l i c Acid 0.032 -S9 50.5 +S9 3.1 i i ) p-Coumaric 5.0 -S9 2.3 Cinnamic Acid +S9 8.4 Caffeic Acid 0.25 -S9 50.1 +S9 3.1 Chlorogenic 0.25 -S9 45.3 Acid +S9 2.2 Flavonoids Quercetin 0.125 -S9 8.0 +S9 1.0 Rutin 2.5 -S9 1.5 +S9 0.0 Catechin 2.5 -S9 8.8 +S9 3.1 Epicatechin 0.8 -S9 24.3 +S9 4.1 Tannins Tannic Acid 0.125 -S9 51.4 +S9 12.4 CHO cel ls were exposed for 3 hours to the phenolic compounds and sampled 20 hours later for chromosome analysis. The concentrations tabulated are the minimum concentrations of test chemical observed to i l l i c i t chromosome damage in a dose response experiment. 39 clastogenic at a dose level as low as 0.032 mg/ml. This activity was abolished by S9. The cinnamic acid, p-coumaric acid with one hydroxyl group, was low in clastogenicity. The addition of S9 increased i t s effect s l ight ly at dose levels of 5.0 mg/ml. Caffeic and chlorogenic acids shared v i r tua l ly the same chromosome damaging effect. Both were active at low doses of 0.063 mg/ml and this effect was abolished when S9 was present. Structurally they are s imilar, with chlorogenic acid containing an additional quinic acid group attached to i t s carboxylic acid group. Ferul ic acid, similar to caffeic acid in structure but with a methyl group substituted for one of the ortho position hydroxyl groups, was s ignif icantly less active. Aberrations were produced at a concentration of 25.0 mg/ml, and these could be eliminated by the addition of S9. In the flavonoid group of phenolics, the flavonol, quercetin, was relat ively active at a concentration of 0.125 mg/ml. This act iv i ty was lost in the presence of S9. Rutin, a flavonol glycoside, was not s ignif icantly active, even at a concentration of 2.5 mg/ml. Because of i t s inso lubi l i ty in aqueous solution, no testing of higher concentrations could be attempted within the experimental guidelines set down in Materials and Methods. The flavan-3-ols, (+)-catechin and (-)-epicatechin, are important components in tannin chemistry. They represent the two most common forms found in plants, of the four possible stereoisomers of the compound. Interestingly, (-)-epicatechin is s ignif icantly more active than (+)-catechin, causing chromosome aberrations at a concentration of 0.8 mg/ml. S9 appeared to decrease this ac t iv i ty . Commercially available tannic acid, which is primarily composed of hydrolysable tannins, was quite active at a concentration of 0.125 mg/ml. The addition of S9 reduced this ac t iv i ty , but did not entirely eradicate i t . 40 Oxidation Effects: The act iv i ty of some of these compounds was found to alter i f the pH of the reaction mixture was allowed to become alkaline before bringing i t back to pH 7.4 or i f the preparation time was increased to several hours or even days. Caffeic and chlorogenic acids may be used as examples of this effect. In Tables 3 and 4, the act iv i t ies of freshly prepared chemical is compared with reaction mixtures allowed to incubate 1 and 3 days at room temperature. These act iv i t ies are also compared with solutions dissolved at pH 10 and allowed to return to pH 7.4 after a 1 hour incubation period at room temperature. The act iv i ty of oxidized solutions was observed to be two to four-fold greater in both cases. This oxidation effect was only observed to occur in phenolics possessing adjacent hydroxy1 groups. That some change had occurred could be readily observed by the dramatic change in colour of the compounds. Figure 4 i l lustrates the change in the v i s ib le absorbance spectrum of caffeic acid solutions at various pH values within 30 minutes of preparation. An aqueous solution of caffeic acid at high pH was observed to change from a colourless to a bright yellow l iquid and f ina l ly to a brown coloured solution with some precipitate. The rate at which these colour changes took place was pH dependent. At higher pH values (pH greater than 12.0) the entire process was complete in about 3 hours. Transition metals were observed to enhance both the colour change and the clastogenic act iv i ty of these compounds. In Table 5, the clastogenic ac t iv i t i es of several phenolics are shown in comparison with their act iv i ty in the presence of copper and manganese metals. In a l l cases of compounds containing adjacent hydroxyl groups, the clastogenic act iv i ty of these compounds was s ignif icantly increased. 41 TABLE 3: The relative clastogenic act iv i ty of" oxidized solutions of Caffeic A c i d . 1 % Metaph ases with Chromosome Aberrations Concentration freshly 1 day 3 day pH altered (mg/ml) prepared solution 2 solution 2 solution 3 2.0 T T T T 1.0 T/MI T/MI T/MI T/MI 0.5 65.9 T/MI T/MI T/MI 0.25 50.0 MI 22.0 MI 0.125 7.5 72.2 23.0 65.4 0.063 3.2 50.0 22.6 32.0 0.032 0.0 14.0 3.0 5.0 0.016 0.0 0.2 1.0 0.0 CHO cel ls were exposed to freshly prepared and autooxidized solutions of caffeic acid for 3 hours and sampled 16 hours later for chromosome analysis. Caffeic acid was prepared as for the freshly prepared solution, at pH 7.0, but allowed to incubate at room temperature for 1 and 3 days. pH altered solutions were prepared by increasing the pH level of the caffeic acid solution to 12.0 for 1 hour at room temperature, before returning i t to pH 7.0. 42 TABLE 4: The relative clastogenic act ivi ty of"" oxidized solutions of Chlorogenic A c i d . 1 Concentration (mg/ml) % Metaph freshly prepared ases with 1 day solution 2 Chromosome Aberrations 3 day pH altered solut ion 2 solution 3 2.0 T T T T 1.0 T/MI T/MI T/MI T/MI 0.5 55.0 T/MI T/MI T/MI 0.25 45.0 MI 18.0 MI 0.125 • •-. 2.1 65.1 22.0 52.1 0.063 0.0 33.2 10.0 17.4 0.016 — 0.0 0.0 0.0 CHO cel l s were exposed to freshly prepared and autooxidized solutions of chlorogenic acid for 3 hours and sampled 16 hours later for chromosome analysis. Chlorogenic acid was prepared as for the freshly prepared solution at pH 7.0, but allowed to incubate at room temperature for 1 and 3 days. pH altered solutions were prepared by increasing the pH level of the chlorogenic acid solution to 12.0 for 1 hour at room temperature, before returning i t to pH 7.0. 43 Figure 4: Caffeic acid absorbance spectrum at various pH values. Curves a, b, c, d, e, and f were generated at pH values of 10.0, 9.0, 8.0, 7.0, 6.0 and 5.0 respectively. A l l solutions were at equal concentration and were measured within 30 minutes of preparation. Solutions at higher pH levels were observed to turn to a bright yellow colour which faded to a dark brown with time. 44 400 500 600 700 Wavelength (nm.) 45 TABLE 5: Relative clastogenic act iv i t ies of phenolics in the presence of transition metals. 1 Phenolic Group Compound Concentration (mg/ml) % Metaphases with Chromosome Aberrations +23 +23 none2 Cu Mn Simple Catechol 0.063 32.0 41.0 75.0 Phenols Resorcinol 2.0 2.0 2.1 2.6 Phenolic p-Hydroxybenzoic 25.0 0.0 0.2 0.0 Acids Acid i) benzoic Gal l i c Acid 0.032 50.5 70.0 T/MI i i ) p-Coumaric Acid 5.0 2.0 1.6 1.0 cinnamic Caffeic Acid 0.25 50.0 60.1 T/MI Chlorogenic 0.25 45.0 61.1 T/MI Acid Ferulic Acid 25.0 10.2 9.8 11.1 CHO cel ls were exposed for 3 hours to the phenolic compounds with or without the transit ion metals present. They were then sampled 20 hours later for chromosome analysis. +2 -4 Cu represents a 10 M f ina l concentration of CuSO^ during the 3 hour incubation period with CHO ce l l s . This concentration was not found to induce chromosome aberrations when treated alone. +2 -4 Mn represents a 10 M f ina l concentration of MnCl^ during the 3 hour incubation period with CHO ce l l s . This concentration was not found to induce chromosome aberrations when treated alone. T/MI is used to describe dose levels of test chemicals which caused toxicity and mitotic inhibit ion in metaphase chromosomes which were scored. 46 This effect was not observed in phenolics with single hydroxyl groups, hydroxyl groups in the meta or para postions with respect to eachother, or with groups possessing methyl substituted hydroxyl groups. Manganese appeared to show a more dramatic enhancing effect than when copper was present. A l l levels of metals tested were within dose ranges -4 not found to cause aberrations by themselves (10 M). Enzymatic Oxidation; Oxidizing enzymes were subsequently used to observe their effect on the induction of chromosomal damage in CHO cel ls by phenolic compounds. Caffeic, chlorogenic, and g a l l i c acids (compounds with two or more adjacent hydroxyl groups) were incubated in the presence of horseradish peroxidase, superoxide dismutase, tyrosinase (monophenol oxidase), and catalase, each at a concentration of 1.0 ug/ml. This concentration was not found to induce genetic damage, when used alone. Table 6 l i s t s the percent cel ls with metaphase chromosomes altered in the presence of these compounds and enzymes. Similar effects were observed with a l l three phenolics. Catalase was observed to reduce some of the toxicity of the three compounds. Its effect was only s l ight , however, in freshly prepared solutions. Superoxide dismutase, and i tyrosinase, on the other hand, appeared to enhance the clastogenic act iv i ty of these compounds. Tyrosinase was observed to change the colour of a l l three compounds to a brown colour within one hour of i t s addition at this concentration. Concurrent application of catalase and superoxide dismutase, both at concentrations of 1.0 ug/ml, was found to reduce the clastogenic ac t iv i t i es of caffeic , chlorogenic, and ga l l i c acids. Table 7 l i s t s the results of this experiment. These act iv i t ies were observed to be below levels measured when only one of the enzymes was present. More TABLE 6: Relative clastogenic ac t iv i t i es of phenolics in the presence of enzymes.1 Concen- % Metaphases with Chromosome Aberrations Compound tration Without Cat. SOD HRP Tyro (mg/ml) Enzyme Caffeic 0.5 T/MI 63.0 T/MI T/MI T/MI Acid 0.25 65.0 31.1 MI 58.0 T/MI 0.125 8.0 6.0 MI 23.0 MI 0.063 3.4 0.0 35.3 5.5 36.1 0.032 0.0 - 23.1 0.0 13.4 Chlorogenic 0.5 T/MI 58.0 T/MI T/MI T/MI Acid 0.25 52.0 50.0 T/MI 45.0 T/MI 0.125 2.8 10.0 MI 20.0 T/MI 0.063 0.0 0.0 10.0 18.0 31.8 0.032 - - 5.0 0.0 9.6 Gal l i c 0.5 T T T T/MI T Acid 0.25 T/MI T/MI T T T 0.125 T/MI T/MI T/MI T/MI T 0.063 MI 38.0 T/MI T/MI T 0.032 51.2 21.2 T/MI T/MI T/MI 0.016 31.6 5.0 MI 38.6 24.6 0.008 0.0 0.0 10.2 15.7 22.1 1 CHO cel ls were exposed to freshly prepared solutions of caffeic, chlorogenic, and ga l l i c acids with and without enzyme for 3 hours. They were then sampled 20 hours later for chromosome analysis. T, and T/MI are used to describe dose levels of test chemicals which caused toxicity and mitotic inhibit ion in metaphase chromosomes which were scored. Abbreviations are as follows: Cat. for catalase, SOD for superoxide dismutase, HRP for horseradish peroxidase, and Tyro, for tyrosinase. A l l enzymes were at a concentration of 1.0 ug/ml, a concentration found not to induce chromosome aberrations when treated alone. 48 TABLE 7: Relative clastogenic act iv i t ies of caffeic acid in the presence of concurrent applications of catalase and superoxide dismu-tase, and catalase with horseradish peroxidase. 1 Compound Concen-tration (mg/ml) % Metaphases Without Enzyme with Chromosome CAT & SOD Aberrations CAT & HRP Caffeic 0.5 T/MI MI MI Acid 0.25 65.0 21.3 64.1 0.125 8.0 4.0 21.0 0.063 3.4 0.0 0.0 0.032 0.0 -Chlorogenic 0.5 T/MI MI MI Acid 0.25 52.0 11.2 32.3 0.125 2.8 3.1 7.8 0.063 0.0 0.1 0.0 0.032 - - -Gal l i c 0.5 T T/MI T/MI Acid 0.25 T/MI MI T/MI 0.125 T/MI 38.9 T/MI 0.063 MI 21.4 34.1 0.032 51.2 7.5 26.2 0.016 31.6 0.0 5.4 1 CHO cel ls were exposed to freshly prepared solutions of caffeic , chlorogenic, and ga l l i c acids, in the presence of catalase with either superoxide dismutase or horseradish peroxidase for 3 hours. They were then sampled 20 hours later for chromosome analysis. The enzymes were present at concentrations of 1.0 ug/ml, a concentration found not to cause aberrations when present alone. Abbreviations are as follows: CAT for catalase, SOD for superoxide dismutase and HRP for horseradish peroxidase. T, and T/MI are used to describe dose levels of test chemicals which caused toxicity and mitotic inhibit ion in metaphase chromosomes which were scored. 49 importantly, much of the toxicity of superoxide-treated samples is reduced by the presence of catalase. Also l i s ted in Table 7 are the results of concurrent application of 1.0 ug/ml each of catalase and horseradish peroxidase. These results appeared very similar to ones produced by catalase alone. It is interesting to note that these enzymes were not sufficient to completely eliminate the clastogenic act iv i ty of the phenolic compounds tested. This would suggest that peroxide and superoxide levels were in excess of 500 uM and 100 uM/min, respectively, in the presence of catalase and superoxide dismutase for caffeic acid, chlorogenic acid and ga l l i c acid. Concurrent application of the enzymes alone was found not to induce chromosome damage. In a separate experiment, caffeic acid that had been allowed to incubate at room temperature for 3 days was tested in the presence of catalase (1.0 ug/ml). Here, catalase completely eliminated i t s act iv i ty on CHO c e l l chromosomes. The results are l i s ted in Table 8. Chromatographic Separation: Oxidation products were chromatographically separated from caffeic acid using high pressure l iquid chromatography (HPLC). Figure 5 demonstrates HPLC profi les of freshly prepared, 1 day, 3 day, and 6 day aqueous solutions of caffeic acid prepared at neutral pH. Here i t can be seen that the main peak at a retention time of 13.8 minutes in the freshly prepared solution diminishes in size with subsequent formation of multiple peaks over the time period examined. Autooxidation appeared to reduce the main peak from greater than 98% of the total solution chromatographed, to approximately 45% of the f ina l to ta l , as judged by absorbance at 260 nm, over a period of 6 days. Samples incubated for the 50 TABLE 8: Relative clastogenic act iv i ty of autooxidized caffeic acid in the presence of catalase enzyme.1 Concentration (mg/ml) % Cells with no catalase Aberrations +catalase 1.0 T 0.5 0.5 22.0 1.0 0.25 23.0 0.5 0.125 22.6 _ 0.063 3.0 — 0.032 1.0 _ 0.016 — — CHO cel ls were exposed to oxidized preparations of caffeic acid with and without catalase enzyme for 3 hours. They were then sampled 20 hours later for chromosome analysis. Caffeic acid was allowed to autooxidize at room temperature at pH 7.0 for 3 days before incubation with CHO ce l l s . Catalase was at a concentration of 1.0 ug/ml, a concentration found not to induce chromosome aberrations. 51 Figure 5: High pressure l iquid chromatography separation of oxidized solutions of caffeic acid. Chromatograms were prepared by measuring the absorbance at 260 nm of products separated on a Vydac 201 TP 5 micron column. Separation was obtained with a 2% methanol per minute gradient starting with 2% methanol, 98% glass d i s t i l l e d water. A l l solvents contained 0.1% phosphoric acid. The solution assayed was a 1 mg/ml preparation of caffeic acid in water, which was allowed to incubate at room temperature for the times specified. Note the reduction of the main caffeic acid peak at 13 minutes, with time. 52 UNOXIDIZEO CAFFEIC ACID O CM < LU O z < CD QC o <n m < 1 DAY OXIDATION 3 DAY OXIDATION ••• M—*-6 DAY OXIDATION - «A A—K—A »_«~—« 1 ' i ' i ' i ' i ' i i i 1 i ' i 1 i 1 I 1 i 1 i 1 l 1 i 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 RETENTION TIME (MINUTES) 53 same period of time, which had been previously purged with nitrogen gas and stored in a ir - t ight containers were not observed to develop any of the oxidation peaks observed above. These oxidation products were chromatographically separated on a Waters preparative reversed phase column, rotary evaporated and tested for clastogenic act iv i ty in the CHO c e l l test system. To verify that these components had not been altered by the extraction procedure, samples. were re-chromatographed on Vydac reversed-phase columns. No apparent degradation could be observed at an absorbance of 260 nm. Overlap of components in this procedure accounted for about 10% of the total peak recovered. Relative concentrations of components to be tested were obtained by calculating the percentage of the total oxidized solution chromatographed, and comparing the areas under the curve obtained for each component. None of the individual components or combinations of major components were observed to exhibit clastogenic act iv i ty even at dose levels 3 times greater than in a 1.0 mg/ml solution. Recombination of a l l components, also fai led to e l i c i t genotoxic effects in the CHO c e l l system at these dose levels. Oxidation Intermediates i) Generation of Peroxide: Oxidation intermediates were measured in a number of ways. was found to be present in oxidized solutions of caffeic acid, using a modified potassium iodide assay. The standard curve for this assay may be found in Figure 2. Here i t can be observed that an increase in absorbance of the potassium iodide mixture correlated l inearly with an increase in H^O^ content in the range of 1 to 100 uM. Caffeic acid's production of ^2°2 may be found graphically represented in Figure 6. During autooxidation a 1 mg/ml solution was 54 Figure 6: The accumulation of I^C^ w i t h time i n a 1 mg/ml s o l u t i o n of c a f f e i c a c i d . H^O> content was assayed i n the potassium i o d i d e assay and p l o t t e d over a d u r a t i o n of 6 days. The c a f f e i c a c i d s o l u t i o n was prepared i n water and allowed to incubate at room temperature f o r the times s p e c i f i e d . 55 1400 1 2 3 4 5 6 7 Time (days) 56 found to contain approximately 60 uM of H^ O^ "" a f t e r 1 day, 180 uM a f t e r 3 days, and 1200 uM a f t e r 6 days. The generation of t^O^ was not s t o i c h i o m e t r i c a l l y r e l a t e d to the disappearance of c a f f e i c a c i d as judged by HPLC separation at an absorbance of 260 nm. Table 9 l i s t s the amount of P r e s e n t at concentrations of c a f f e i c a c i d tested i n the CHO c e l l assay. A p a t t e r n of increased c l a s t o g e n i c a c t i v i t y was found to p a r a l l e l the e a r l y process of au t o o x i d a t i o n i n c a f f e i c a c i d , however, t h i s was followed by a d e c l i n e i n a c t i v i t y i n the 3 day and 6 day s o l u t i o n s . That m u s t P^ a v some r o l e i n the observed a c t i v i t y i s s u b s t a n t i a t e d by a l o s s of a c t i v i t y i n 3 day s o l u t i o n s upon the a d d i t i o n of a s o l u t i o n of 1.0 ug/ml ca t a l a s e (Table 10). Table 11 documents the c l a s t o g e n i c a c t i v i t y of a stock ^2^2 so^-ution. Here i t may be seen that a s i m i l a r p a t t e r n of chromosome damage can be produced w i t h concentrations of 10 to 50 uM ^2^2' l e v e l s found i n a 3 day s o l u t i o n of c a f f e i c a c i d . ^02 does not, however, appear to account f o r the a b i l i t y of f r e s h l y prepared c a f f e i c a c i d to damage chromosomes. i i ) Generation of Superoxide: The production of another o x i d a t i o n i n t e r m e d i a t e , the superoxide or 0^' f r e e r a d i c a l , was measured using a ferricytochrome c assay. This assay measures the change i n absorbance at 550 nm of a ferricytochrome c mixture as i t changes from i t s o x i d i z e d to i t s reduced form. Superoxide dismutase was used to measure any background changes. In Figure 7, the production of 0^' can be found i l l u s t r a t e d at pH values of 7.0, 8.0, and 9.0. The rate of production of 09" i s pH 57 TABLE 9: Hydrogen peroxide content of freshly prepared and autooxidized caffeic acid. 1 H 0 2 Content (uM) Concentration freshly 3 day (mg/ml) prepared solution 1.0 17.0 180 0.5 9.0 90 0.25 4.0 45 0.125 2.0 22 0.063 1.0 11 0.032 <1.0 6 0.016 <1.0 3 0.008 <1.0 1 Hydrogen peroxide content was determined using a modified potassium iodide assay on the concentrations of caffeic acid l i s t ed . A l l values were obtained using the standard curve outlined in Figure 2. Autooxidized solutions were prepared by making up solutions of caffeic acid at pH 7.0 and allowing them to incubate at room temperature for a period of 3 days. 58 TABLE 10: The clastogenic act iv i ty of freshly prepared and autooxidized caffeic a c i d . 1 % Co11 s with Aberrations Concentration freshly prepared 3 day solution (mg/ml) no catalase +catalase no catalase +catalase 1.0 T/MI T/MI T 1.0 0.5 12.3 8.2 T 0.5 0.25 3.8 1.0 MI _ 0.125 1.0 — MI _ 0.063 0.5 — 18.4 _ 0.032 — 1.0 -CHO cel ls were exposed to freshly prepared and 3-day oxidized solutions of caffeic acid, with or without catalase enzyme present, for 3 hours. They were then sampled 20 hours later for chromosome analysis. Catalase was used at a concentration of 1.0 ug/ml, a concentration found not to induce chromosome aberrations when present alone. 59 TABLE 11: The clastogenic act iv i ty of commercially available hydrogen peroxide. 1 Peroxide % Cells with Aberrations Concentration no catalase •catalase (pM) 500 T 1.0 250 T 0.5 125 MI _ 62 MI _ 31 18.4 _ 16 1.0 _ 8 — _ 4 — -CHO cel ls were exposed for 3 hours to commercially available hydrogen peroxide and sampled 3 hours later for chromosome aberrations. Catalase was present at a concentration of 1.0 yg/ml, a concentration found not to induce chromosome aberrations when present alone. 60 Figure 7: The rate of production of 0^ in a 1 ml aliquot of a 10 M caffeic acid solution at various pH values. Curve "a" was generated by adding caffeic acid to a stock solution of ferricytochrome c at a f ina l pH level of 8.97 and monitoring the change in absorbance at 550 nm. Curves "b" and "c" were prepared in an identical fashion but were added to buffered solutions of ferricytochrome c at f i n a l pH values of 7.98 and 6.99, respectively. The rate of production of O2" was calculated at half_^maximum absorbance, using a molar extinction coefficient of 21 mM cm 61 .07' Time (minutes) 62 dependent, being greatest at the higher pH v a l u e s . Using a molar e x t i n c t i o n c o e f f i c i e n t of 21 mM * cm ^ (Van Gelder and S l a t e r , 1962) the r a t e of production of C^' can be c a l c u l a t e d using the f o l l o w i n g formula: AAbs. x 1 = conc e n t r a t i o n of = KQ ^ t 21 ferricytochrome c 2 at h a l f maximum where AAbs.= change i n h a l f maximum absorbance at 550 nm t = time ( i n minutes) at which h a l f maximum absorbance was reached. KQ was c a l c u l a t e d to be approximately 1.42 uM/min at pH 7.0, 2.86 uM/min at pH 8.0 and 3.81 uM/min at pH 9.0 f o r a l x l O - 4 M s o l u t i o n of c a f f e i c a c i d . These values are expressed i n terms of r a t e of production of 0^' at one h a l f the maximum change i n absorbance. i i i ) Generation of Phen o l i c Free R a d i c a l s : Aside from the production of intermediate 1^02» and O2" > the parent p h e n o l i c molecule was a l s o found to undergo a t r a n s i t i o n to a fre e r a d i c a l s t a t e . This could be most e a s i l y v i s u a l i z e d by absorbance wavelength scans of c a f f e i c and g a l l i c a c i d s at higher pH value's (pH 9.0), or by o x i d a t i v e degradation w i t h s e l e c t e d enzymes. Figure 8 demonstrates the change i n the abso r p t i o n spectrum of a -4 10 M s o l u t i o n c a f f e i c a c i d at pH 9.0 over a 30 minute i n t e r v a l . Wavelength maximums at approximately 280 and 315 nm were found to degenerate w i t h a new maximum forming at 415 nm. An almost i d e n t i c a l 63 Figure 8; The change in absorbance in caffeic acid at a pH of 9.0. The straight l ine indicates the absorbance spectrum of an aqueous solution of caffeic acid (pH 9.0) immediately after the preparation. The dashed l ine i l lus trates the spectrum of the same solution at pH 9.0, 30 minutes after preparation. 64 250 350 450 550 Wavelength (nm.) 65 profi le (Figure 9) was generated by the addition ""of 33 ug/ml horseradish -2 peroxidase and 10 M H2^2* N o t e that no oxidation took place unt i l both the enzyme and the peroxide were present. Figures 10 and 11 show similar results for ga l l i c acid. A wavelength maximum at 260 nm was observed to degenerate with subsequent production of a new peak at 415 nm at high pH (9.0) and by enzymatic oxidation with horseradish peroxidase. To identify i f phenolic free radical molecules were present, electron spin resonance (ESR) was performed on several of the phenolic molecules under a variety of conditions. As was expected, no ESR signal could be detected for the monohydroxy phenols, m- and p-coumaric, and ferul ic acids even under conditions of high pH (pH greater than 12.0) and maximum input power (100 mWatts or mW) (Figure 12). The phenolics possessing adjacent hydroxyl groups, however, generated dist inct signals under the conditions tested. Figure 12 i l lustrates the primary phenolic free radical of protocatechuic acid, or 2,3-dihydroxybenzoic acid. This signal was generated with 1 mW of microwave power and had a gain value of 1.25 x 10 . It has 8 dist inct hyperfine structures, 4 arranged on each side of a symmetry point. The pattern is characteristic of a molecule with 3 unequal interacting nuclei , each with a spin of 0.5. The g value may be calculated to be 2.0042 ± 0.0002 based on the g-value of 2.0036 ± 0.0002 for DPPH ( K i t t e l , 1971). Values of hyperfine coupling constants for protocatechuic acid are given below. These were obtained by comparing the separation of peaks in terms of gauss distribution with published values. Complete overlap of published values with ones recorded in this study would indicate that hyperfine constants were completely analogous to the f i r s t free radical derived from oxidation of protocatechuic acid. 66 Figure 9: The change in absorbance of caffeic acid when incubated with a solution of 33 ug/ml horseradish peroxidase and 10 M ^2^2' a t ^ 7.0. The straight l ine indicates the absorbance spectrum of an aqueous solution of caffeic acid at pH 7.0. The dashed l ine is the curve generated 30 minutes after the addition of peroxide and peroxidase enzyme. Addition of peroxide or peroxidase enzyme alone was not found to change the absorbance spectrum from the one measured at pH 7.0 alone. 67 Absorbance (O.D. oo In 1st O Figure 10; The change in absorbance of ga l l i c acid at a pH of 9.0. The straight l ine indicates the absorbance spectrum of an aqueous solution of ga l l i c acid, pH 9.0, immediately after preparation. The dashed l ine i l lustrates the spectrum of the same solution at pH 9.0, 30 minutes after preparation. 69 i.oH j i i i — 250 350 450 550 Wavelength (nm.) 70 Figure 11; The change in absorbance of g a l l i c acid when intubated with a solution of 33 ug/ml horseradish peroxidase and 10 M H2^2' a t ^ 7.0. The straight l ine indicates the absorbance spectrum of an aqueous solution of ga l l i c acid at pH 7.0. The dashed l ine is the curve generated 30 minutes after the addition of peroxide and peroxidase enzyme. Addition of peroxide or peroxidase enzyme alone was found not to change the absorbance spectrum from the one measured at pH 7.0 alone. 71 -1 250 350 Wavelength (nm.) t 450 550 72 Figure 12: ESR spectra of 10 solutions of "a" (m-coumaric acid) "b" (p-coumaric acid) "c" (ferul ic acid) and "d" (protocatechuic acid) at pH 12.0. Measurements were made at 100 mW of power for "a", "b", ajjd "c", and at 1 mW of power for "d". The gain setting was 1.25 x 10 , with a modulation amplitude of 0.2 G, and a time constant of 0.25 seconds at a f i e ld setting of 3294 G, and microwave frequency of 9.45 GHz. A l l measurements were at room temperature and modulation frequency of 100 KHz. Signal for "a", "b", and "c" could be observed even at higher gain settings of 1.25 x 10 . 73 Hyperfine c o u p l i n g constants of protocatechiric a c i d : C O Q H Hyperfine Coupling (G) pH a, H H H *2 5 12.0 0.5 3.5 0.9 from: Kalyanaraman and Sealy, 1982, Figure 13 represents the phenolic free radical signal of ga l l i c acid at pH 12.0. This signal was relat ively strong and could be detected with a receiver gain setting of only 5.0 x 10 at 1 mW of microwave power. The pattern obtained is dist inct ive and may be referred to as a 1:2:1 hyperfine structure based on the ratio of i t s peak heights. The ESR hyperfine pattern of ga l l i c acid is consistent for a molecule with 2 equal nucle i , each with a spin value of 0.5. The g-value calculated for ga l l i c acid was 2.0049 ± 0.0002 based on the g-value of 2.0036 for DPPH ( K i t t e l , 1971). At pH 7.75, no signal could be detected (Figure 14) even at a 4 receiver gain level of 1.25 x 10 . This was true even in samples allowed to autooxidize at room temperature for several days. However, -2 the addition of 50 ug/ml horseradish peroxidase and 10 M ^ 2^2 t 0 freshly prepared g a l l i c acid, led to the generation of the dist inct ive 1:2:1 hyperfine pattern. The signal generated in Figure 15 was considerably weaker than the one generated at high pH, and required Figure 13: ESR spectra of "a" (10 _ 2M g a l l i c acid at pH 12.0) and "b" (crystal l ine diphenylpicrylhydrazyl or DPPH). Measurements were made at 1 mW ojj microwave power, with a gain of 50, modulation amplitude of 3.2 x 10 G, time constant of 1.0 second at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and modulation frequency of 100 KHz. 76 77 Figure 14: ,-2 ESR spectra of 10 ' H solutions of "a" (freshly prepared g a l l i c acid at pH 7.75) and "b" (gal l ic acid at pH 7.75 after 3 days). Measurements were made at 20 mW of microwave^ power, with a gain of 1.25 x 10 , modulation amplitude of 4.0 x 10 G, time constant of 1.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and modulation frequency of 100 KHz. 78 Figure 15: ESR spectra of 10 ^ M solutions of ga l l i c acid at pH 7.75. Figure "a" is the signal generated in the absence of enzyme and figure "b" is the signal obtained upon addition of 33 ug/ml horseradish peroxidase and 10 M ^^^2' Measurements^ were made at 20 mW of microwave^ power, with a gain of 1.24 x 10 , modulation amplitude of 4 x 10 G, time constant of 1.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and a modulation frequency of 100 KHz. 80 receiver gain levels of 1.25 x 10^. Some of the"structure of the third peak appears lost in the background noise at this level . An analogous structure was derived by the addition of tyrosinase to a freshly prepared solution of ga l l i c acid. It is represented in Figure 16. In the case of ga l l i c acid, values for hyperfine coupling constants were observed to be identical for signals generated by high pH and by the enzymes, peroxidase and tyrosinase. Values were assigned by comparing the separation of peaks observed in this study with those of published values. The hyperfine constants of ga l l i c acid are given below: C O O H Hyperfine Coupling (G) „ H H pH 12.0 0.10 0.10 7.5 a 0.10 0.10 a: in the presence of peroxidase and tyrosinase as outlined in Materials and Methods. from: coupling constants calculated by Dixon and Murphy, 1975. Figure 17 represents the phenolic free radical signal of caffeic acid at pH 12.0. Again this signal was relat ively strong and could be 3 detected at a receiver gain level of 8 x 10 at 1 mW of microwave power. This pattern is exceedingly complex and is approximately what 82 Figure 16: ESR spectra of 10 ^ M solutions of g a l l i c acid at pH 7.75. Figure "a" is the signal generated in the absence of enzyme and figure "b" is the signal obtained upon addition of 50 ug/ml tyrosinase (monophenol oxidase). Measurements were made at 20 mW of microwave powder, with a gain of 1.24 x 10 , modulation amplitude of 4 x 10 G, time constant of 1.0 sec at a f i e l d setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and amodulation frequency of 100 KHz. 83 Figure 17; ESR spectra of "a" (crystall ine DPPH) and "b" (10 M caffeic acid at pH 12.0). Measurements were made a 1 mW of microwave power, with a gain of 8 x 10 , modulation amplitude of 8 x 10 G, time constant of 3.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and a modulation frequency of 10 KHz. 85 86 one would expect for a molecule with 5 unequal nuclei , each with a spin value of 0.5. Twenty nine of the 32 expected peaks (2 5 = 32) are resolved. If one alters the modulation amplitude to a setting of 8 x 10 \ and the time constant to 1.0 second, one is able to observe the main 7 peaks underlying i t s structure (Figure 18). This signal prof i le is important when comparing spectra of enzymatically-derived free radicals of caffeic acid. At pH 7.5, no signal could be detected even at maximum receiver gain level . The addition of 50 yg/ml horseradish peroxidase and 10 M 1^ 02 led to the generation of the signal i l lustrated in Figure 19. This signal is considerably weaker and required maximum gain levels of 4 2.5 x 10 . Inherent in this spectra are the same 7 main peaks observed at alkaline pH. This was also observed to be the case when the enzyme, tyrosinase, was present (Figure 20). Using peak separation diagrams of published values, hyperfine coupling constants were obtained and are l i s ted below for caffeic acid. CH^CHCOOH a 6 Hyperfine Coupling (G) u H H H pH a 0H c a,H a D v 2 a5 6 aa 8 12.0 0.023 0.119 0.282 0.130 0.236 7.5 3 0.022 0.119 0.283 0.130 0.237 a: in the presence of peroxidase and tyrosinase, as outlined in Materials and Methods. coupling constants calculated from: Dixon et a l . , 1975; Ashworth, 1976. 87 Figure 18: ESR spectra of a 10 M solution of caffeic acid at pH 12.0. The measurement was made made at 1 mW of microwave power, with a gain of 2.5 x 10 , modulation amplitude of 8 x 10 G, time constant of 1.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and a modulation frequency of 100 KHz. 88 89 Figure 19: ESR spectra of 10 M solutions of caffeic acid at pH 7.5. Figure "a" is the signal generated in the absence of enzyme and figure "b" is the signal obtained upon addition of 33 ug/ml horseradish peroxidase and 10 M H^O^. Measurements ^were made at 20 mW of microwav^ power, with a gain of 1.24 x 10 , modulation amplitude of 4 x 10 G, time constant of 1.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz-. A l l measurements were made at room temperature and a modulation frequency of 100 KHz. 90 Figure 20: ESR spectra of 10 ^ M solutions of caffeic acid at pH 7.5. Figure "a" is the signal generated in the absence of enzyme and "b" is the signal obtained upon addition of 50 ug/ml tyrosinase (monophenol oxidase). Measurements were made at 20 mW of microwave powpr, with a gain of 1.24 x 10 , modulation amplitude of 4 x 10 G, time constant of 1.0 sec at a f i e ld setting of 3355 G and microwave frequency of 9.45 GHz. A l l measurements were made at room temperature and a modulation frequency of 100 KHz. 92 Here we can observe the s i g n i f i c a n t c o n t r i b u t i o n of the cinnamic s i d e chain i n generating complexity of s i g n a l . 94 DISCUSSION Phenolics and the Environment: It has been estimated that most human cancers are of an environmental origin (for reviews see Higginson, 1968; Higginson and Muir, 1973; Armstrong and Do l l , 1975; Wynder, 1976; Wynder and Gori , 1977). Although some of these cancers are due to occupational exposure to carcinogens and to smoking, a larger proportion can be correlated with dietary patterns. Table 12 l i s t s proportions of cancer deaths attributable to various environmental factors, as reported by Dol l & Peto in their report on "Avoidable Risks of Cancer in the U.S." commissioned by the U.S. Congress in 1981. Here i t may be seen that diet plays a significant role . The plant phenolics, which comprise the subject matter of this thesis, constitute a major portion of the components consumed in man's daily diet . Estimates vary but a generally accepted range is between 600 mg and several grams per day (Powell et a l . , 1974; Maga, 1978). Their bio logical act iv i ty is therefore of some interest in determining the role they may play in human carcinogenesis. Because of the great diversity of phenolic substances and the wide range of experimental conditions used, confl ict ing reports have been published on their role in carcinogenesis. The synthetic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, have both been reported to inhibit carcinogen-induced neoplasia (Speier et a l . , 1978; Wattenberg, 1972; Wattenberg, 1973; Wattenberg et a l . , 1979). More recently, Wattenberg et a l . , 1980, have reported that the phenolic derivatives of cinnamic acid, such as O-hydroxycinnamic acid, 3,4-dihydroxy-cinnaraic acid, were also able to inhibit benzo(a)pyrene-induced neoplasia in mice. 95 TABLE 12: Proportions of cancer cases attributed to various different factors by different authors. Percent of a l l cancer cases in: Factor or class of factors England.Birming-ham region,based on Higginson and Muir (1979) United States, based"on Wynder and Gori (1977)* Male Female Male Female Tobacco 30 7 28 8 Tobacco/alcohol 5 3 4 1 Diet — — 40 57 Li fe - s ty le 30 63 — — Occupation 6 2 4 2 Sunlight 10 10 8 8 Ionizing radiations 1 1 Iatrogenic 1 1 — — Exogenous hormones — — — 4 Congenital 2 2 16 20 Unknown 15 11 * Deduced from histograms, congenital and unknown. Non-environmental factors equated with From, Dol l and Peto, 1981, p. 1257. 96 In a review art ic le by Stich and Rosin, 1983, phenolic derivatives were reported to inhibit the mutagenic ac t iv i t i es of both direct-acting carcinogens and precarcinogens in the presence of mixed function oxidases. The report also goes on to demonstrate that they are able to reduce the in v i tro formation of mutagenic and carcinogenic N-nitroso compounds. In man, phenolics were observed to reduce levels of nitrosoproline in urine, following administration of ni trate , proline and phenolic test substances. Many deleterious effects have also been reported. Table 13 is a composite l i s t of genotoxic events caused by phenolics under a variety of test conditions. Not included are the more obvious effects known to be brought about by the carcinogens, Aflatoxin Bl and safrole. For reviews of the more complex group of flavonoids see Nagao et a l . , 1981, and Brown, 1980. The Induction of Chromosomal Abnormalities by Phenolics: In Table 2 i t may be observed that several of the phenolics tested caused chromosomal abnormalities in a CHO c e l l test system. These results para l l e l findings by Stich et a l . , 1981a. Two aspects are dist inct ive to this table and the results reported by Stich et a l . , 1981a. The f i r s t pattern is the more extreme effect of phenolics possessing adjacent hydroxyl groups, and the second pattern is that the presence of S9 diminished most clastogenic act iv i ty . Because molecules with adjacent hydroxyl groups are much more susceptible to oxidation, an investigation was begun on their a b i l i t y to oxidize and form free radical by-products. It was believed that the oxidation products were responsible for the observed clastogenic act iv i ty and that the presence of S9 might serve to detoxify or modify such reactions at higher pH values. TABLE 13 : Genotoxic e f f e c t s measured w i t h p l a n t phenolics PHENOLIC GROUP ASSAY ORGANISM REFERENCE 1. SIMPLE PHENOLS phenol s i s t e r chromatid exchange human lymphocytes Morimoto and Wolff (1980) cat e c h o l p o i n t mutation m i t o t i c crossover s i s t e r chromatid exchange n e p h r o t o x i c i t y chromosome aberrations S. c e r e v i s i a e S. c e r e v i s i a e human lymphocytes r a t s CHO c e l l s Kunz et a l . (1980) Kunz et a l . (1980) Morimoto and Wolff (1980) Calder e t a l (1975) S i t c h e t a l (1981a) p y r o g a l l o l p o i n t mutation p o i n t mutation m i c r o n u c l e i chromosome aberrations S. typhimurium E. c o l i mice CHO c e l l s Ben-Gurion (1979) Yamaguchi (1981) B i l i m o r i a (1975) M i t r a and Manna (1977) Gocke et a l . (1981) S t i c h et a l . (1981a) r e s o r c i n o l DNA i n h i b i t i o n m i c r o n u c l e i mice mice S e i l e r (1977) M i t r a and Manna (1977) 2. BENZOIC ACIDS g a l l i c a c i d p o i n t mutation chromosome aberrations S. typhimurium CHO c e l l s Yamaguchi (1981) S t i c h et a l . (1981a) pro t o c a t e c h u i c a c i d chromosome aberrations CHO c e l l s S t i c h et a l . (1981a) v a n i l l i c a c i d chromosome aber r a t i o n s CHO c e l l s S t i c h et a l . (1981a) continued . . . TABLE 13 continued: 2. CINNAMIC ACIDS c a f f e i c a c i d c h l o r o g e n i c a c i d gene conversion chromosome aberrations gene conversion chromosome aberrations . c e r e v i s i a e S t i c h and Powrie (1982) CHO c e l l s S t i c h et a l . (1981a) . c e r e v i s i a e S t i c h et a l . (1981a) CHO c e l l s S t i c h e t a l . (1981b) i The Oxidation of Phenolics: The oxidation of phenolic molecules was observed to occur more rapidly at alkaline pH. Caffeic, chlorogenic and ga l l i c acids were a l l observed to change dramatically in so lubi l i ty and colour as their pH level was increased. Figure 4 i l lus trates the colour changes in caffeic acid. This effect is not surprising i f we examine the reaction mechanisms outlined in Figure 21. The free radical resonance structures generated by high pH levels may be seen to form dimers with other radicals . This involves the formation of new C - C , C - 0, or 0 - 0 bonds (in order of decreasing importance) which may be followed by further rearrangements. Continued oxidation produces polymeric material and many complex dark-coloured materials often synthesized by plants (Thomson, 1964). Phenols with monohydroxy or hydroxyl groups in the meta or para positions are much slower to oxidize. This is because they lack the resonance s tabi l izat ion process outlined in part A of Figure 21. Without this degree of resonance s tabi l izat ion , the phenoxy free radical is less l ike ly to be formed, and once formed, is much shorter l ived. The change of co l l i s ion and bond formation with another free radical is therefore much diminished. HPLC separation of caffeic acid and i t s oxidation products using a reversed-phase Vydac column and a methanol gradient, demonstrated the presence of many oxidation products (Figure 5). On the basis of ul traviolet absorption at 260 nm, greater than 96% of a freshly prepared aqueous solution of caffeic acid was present as the parent compound. However, when the compound was prepared in i t s salt form at pH 7.00 for biological experiments, there was observed to be a 5 - 10% degradation of the compound. After a 6 day incubation period, only 45% of the orig inal caffeic acid peak remained. The rest was present as oxidative 100 Figure 21; Resonance s tabi l izat ion and dimer formation in phenols with hydroxyl groups in the ortho posit ion. Figure 21A i l lustrates some of the resonance s tabi l izat ion structures available to the molecule. From R.H. Thomson, 1964, p. 16. 101 21 A 102 by-products of various molecular weights. The Characterization of Phenolic Oxidation Products: If oxidation does play a major role in the genotoxicity of phenolics, i t becomes important to isolate and characterize by-products which contribute to the act iv i ty of such a reaction. Employing a Waters reversed phase preparative column, high pressure l iquid chromatography was used to separate out each of the component oxidation products of caffeic and chlorogenic acids. Samples were concentrated by rotary evaporation and tested in the CHO c e l l test system. None of the components tested could account for the clastogenic act iv i ty of the parent compound, even when tested at concentrations 3 times those found in a 1.0 mg/ml solution. Various combinations of peaks, and total recombination of peaks, also fai led to induce clastogenic act iv i ty . It would therefore appear that the source of clastogenic act iv i ty must either reside in the front peak of the chromatographic separation (not retained on a reversed-phase column) or was relat ively unstable and did not survive the chromatographic separation and subsequent rotary evaporation. In an effort to resolve which was the case, tests were undertaken to identify and characterize oxidation products not retained on chromatographic columns. Based on the chemical structure of these compounds, i t was hypothesized that one such by-product could be hydrogen peroxide. Testing with a potassium iodide assay for the presence of peroxide, demonstrated significant levels of hydrogen peroxide in solutions of caffeic acid that had been allowed to autooxidize at room temperature over a period of days (Table 9). Elevated levels of hydrogen peroxide were also observed to occur in solutions raised in pH level for a period of at least one hour before returning to pH neutrality. 103 The production of hydrogen peroxide was observed to occur at the same time as an increase in clastogenic act ivi ty of 1 day oxidized solutions. This act iv i ty appeared to taper off in solutions incubated for greater than 3 days. Tables 3 and A i l lus trate this effect for both caffeic and chlorogenic acids. Hydrogen peroxide may be scavenged by two classes of related enzymes - the catalases and the peroxidases. These enzymes catalyze the divalent reduction of ^^0^ to 2 ^ 0 , ' using 2^^ 2 a s t ' i e e l e c t r o n donor in the case of catalases, or using a variety of reductants in the case of peroxidases (Fridovich, 1976). Commercially prepared catalase was observed to eliminate a l l clastogenic act iv i ty in both autooxidized solutions of caffeic acid and in hydrogen peroxide treated solutions (see Tables 10 and 11). That catalase had in fact removed a l l 2^^ 2 W a S v e r i f i e d with the potassium iodide colourimetric assay. The hydrogen peroxide solutions were tested at concentrations found to be present in oxidized solutions of caffeic acid (Table 9). A similar degree of act iv i ty was observed in the 10 to 50 uM range for both cases. This would allow one to infer that some portion of the oxidized caffeic acid's act iv i ty was due to the presence of hydrogen peroxide. In CHO c e l l cultures exposed to caffeic acid in the presence of horseradish peroxidase, the results were less dramatic (Table 6). Freshly prepared solutions did not appear to possess sufficient H^O^ to catalyze and carry on the reaction, while oxidized solutions appeared to have polymerized to a point that there was a general inabi l i ty by the phenolics to act as reductants. Consequently, there was only a slight ( i f any) reduction in the clastogenic act iv i ty of caffeic acid in the presence of this peroxidase. This result has also been reported for ga l l i c acid by Kamel et a l . , 1977. 104 Concurrent application of both catalase and peroxidase in freshly prepared solutions of caffeic acid produced no observed effect on the level of act iv i ty of caffeic acid on CHO c e l l chromosomes (Table 7). Presumably, the catalase destroyed any 1^ 02 present, therebye interfering with the in i t i a t ion of the peroxidase-catalyzed reaction. Further evidence of peroxide involvement comes from the measurement of enhanced act iv i ty in the presence of transition metals. Marte l l , 1980, has shown that compounds of a similar structure in the presence of transition metals, produce hydrogen peroxide. Figure 22 i l lus trates a possible mechanism of the catalyt ic effect of manganese (Mn) during the oxidation of caffeic acid (modified from Marte l l , 1980). Studies in this laboratory using HPLC (unpublished) have indicated that manganese does indeed stimulate the oxidative degradation of caffeic and chlorogenic acids at neutral pH. The generation of peroxide during this accelerated oxidation may account for the increased biological act iv i ty of some phenolic compounds in the presence of transition metals, such as Mn. In Table 10 i t may be observed that catalase was able to eliminate the clastogenic act iv i ty of oxidized solutions but not freshly prepared solutions of caffeic acid. This observation would lend support for the hypothesis of another oxidative by-product. One such product was measured to be superoxide or O2' Using a modified ferricytochrome c assay, elevated levels of 0^' were measured at pH values of 7.0, 8.0 and 9.0 (Figure 7). These levels were completely eliminated by the addition of superoxide dismutase. The rate of production of O2' was observed to be pH- dependent. Rates of production of superoxide anion radical were observed to be 1.42 uM/min at pH 7.0, 2.86 uM/min at pH 8.0, and 3.81 uM/min at pH 9.0 -4 for a 1 x 10 M solution of caffeic acid. These values are 105 Figure 22: Manganese catalysis of peroxide formation during the oxidation of a dihydroxyphenol. This is a proposed mechanism to i l lus trate the enhancing effect of transit ion metals on the clastogenic act iv i ty of plant phenols. Modified from A . E . Marte l l , 1980, p. 95. 106 Generation of H 2 0 2 ! 107 higher than rates published by Aver'yanov, 1981. For a 1 x 10 H M solution of caffeic acid, they measured rates of 0.073 pM/min at pH 7.0 and 0.105 pM/min at pH 8.0. One reason for this discrepancy is that they use a different molar extinction coefficient for ferricytochrome c. Their value is l i s ted as 18.5 mM * cm * from Margoliash and Frohwirt, 1959. The value used in this study was 21 mM * cm ^ (from Van Gelder and Slater, 1962), a difference of approximately 8%. It is not shown how the rates of production of superoxide are calculated in the Aver'yanov paper, but the overall change in absorbance during the reduction of ferricytochrome c appears to be similar. Perhaps i t is sufficient to say that they were able to observe the production of superoxide anion radicals from caffeic acid. A possible mechanism for the production of O2* during the oxidation of caffeic acid is outlined in Figure 23. Because of i t s transitory nature, one would not expect superoxide free radicals to play a role in completely oxidized solutions of phenolics. It would seem plausible that the i n i t i a l peak of clastogenic act iv i ty at 1 day of incubation, might be due to the presence of these short-lived free radicals . However with the subsequent formation of the polymerized quinone structures (Thomson, 1964) their e lectrophil ic structure may act as scavengers of free radical products, and at least account in part for the sl ight decrease in act iv i ty of 3 day and 6 day samples. The addition of superoxide dismutase to CHO c e l l cultures containing caffe ic , chlorogenic or g a l l i c acids appeared to increase the genotoxicity of these compounds. This result is not surprising i f we examine the enzyme mechanism as outlined by McCord and Fridovich, 1969. 2 0 2 T + 2H+ -> 0 2 + H 2 0 2 108 Figure 23; Proposed mechanism f o r the production of superoxide anion f r e e r a d i c a l s from dihydroxyphenpls i n the presence of a l k a l i . T his i s a r e a c t i o n which can only take place under aerobic c o n d i t i o n s . 109 Generation 110 The net conversion of C^" to would therefore account for increased toxicity due to the accumulation of the more stable product, hydrogen peroxide. One would suspect concurrent applications of superoxide dismutase and catalase to decrease the genotoxicity of freshly prepared solutions of caffeic, chlorogenic and ga l l i c acids. Table 7 i l lus trates that this i s the case. The effect, however, is not total and several reasons may be put forth to explain this . The f i r s t and most obvious explanation is that the enzymes were not in sufficient concentration to eliminate a l l the H2O2 and O2* generated. Normally, this should not be the case for catalase, because the concentration used (5ug/ml) was shown to be sufficient to remove a l l clastogenic act iv i ty of 500 uM solutions of H2O2. The level of superoxide dismutase used (1.0 ug/ml) was found to be sufficient to catalyze the conversion of 100 uM of 0^' /min to H2O2 in the xanthine/xanthine oxidase generating system. It seems doubtful that the rate of production of O2* from the oxidation of caffeic acid would exceed this value. Kono and Fridovich, 1982, have recently published a paper demonstrating that O2" can actually reversibly inhibit the ab i l i t y of catalase to breakdown 2^^ 2* m a ^ therefore be possible that active production of O2* by the oxidizing caffeic acid molecules may be inhibit ing catalase in i t s function. The transitory nature of the O2" radical would suggest that this is only a temporary effect. This consideration would therefore only play a minor role in catalase mechanics after a 1 hour pre-incubation period. Another possible explanation is that there are s t i l l other oxidation products present which are capable of inducing genetic damage. Several factors point to this conclusion. The enzyme tyrosinase is an oxygen and four electron transferring monophenol oxidase present in 111 almost a l l b i o l o g i c a l systems. I t ca t a l y z e s the o x i d a t i o n of s e v e r a l phenolics w i t h the net production of l^O; but no or 0^'. This r e a c t i o n as o u t l i n e d i n the Worthington Enzymes Catalogue and i s given below. When ty r o s i n a s e was added to aqueous suspensions of c a f f e i c , chlorogenic and g a l l i c a c i d s at n e u t r a l pH, they were observed to s o l u b i l i z e and change q u i t e d r a m a t i c a l l y i n colour and f i n a l l y to a dark brown. No ^ C ^ could be detected using the potassium i o d i d e assay. When CHO c e l l s were incubated w i t h c a f f e i c , chlorogenic and g a l l i c a c i d s , i n the presence of t y r o s i n a s e , the c l a s t o g e n i c a c t i v i t y of these compounds was n o t i c e a b l y increased (Table 6 ). One p o s s i b l e e x p l a n a t i o n , then, f o r the a c t i v i t y observed i n the superoxide dismutase/catalase experiment and i n the t y r o s i n a s e treatment experiment, would be the presence of phenolic f r e e r a d i c a l s . These f r e e r a d i c a l s would act as intermediates i n quinone formation and would be expected to be r e l a t i v e l y short l i v e d . One would ther e f o r e not a n t i c i p a t e such molecules to play a r o l e i n the c l a s t o g e n i c a c t i v i t y of 3 day o l d s o l u t i o n s of p h e n o l i c s . Peroxide appears to be the genotoxic agent at that time. They would, however, be present i n f r e s h l y prepared s o l u t i o n s which were a c t i v e l y t a k i n g part i n the o x i d a t i o n process. Although there i s no d i r e c t way of assaying t h e i r a b i l i t y to induce genetic damage, t h e i r presence can be detected by the use of e l e c t r o n s p i n resonance measurements. 112 Electron Spin Resonance of Phenolic Free Radicals': The presence of an odd electron generated during the oxidative process, makes electron spin resonance the method of choice in studying phenolic autooxidation. As expected, phenolic molecules which lacked clastogenic act iv i ty in the CHO c e l l chromosome aberration tests also fai led to generate a signal even under conditions of high pH. Figure 12 i l lustrates that this is the case. These molecules could be classif ied as those having one or more hydroxyl groups in the - meta or para positions with/or without a modified adjacent hydroxyl group (for example, M-coumaric, p-coumaric and ferul ic acids). It should be pointed out that these phenolic acids are capable of autooxidation, but that the reaction rate was so slow as to make the generation of free radicals' signals too weak to be detected. Phenolic acids which were observed to be active in the CHO c e l l test also appeared to generate strong ESR signals under oxidative conditions. The best example, ga l l i c acid or 3,4,5,-trihydroxybenzoic acid i l lustrates this point. This molecule has three adjacent hydroxyl groups capable of forming semiquinone and quinone structures. With the presence of three hydroxyl groups adjacent to each other there occurs a fa i r degree of resonance stabi l izat ion and the signal generated by this molecule is re lat ively strong. The odd electron which is generated during the oxidation process is then free to interact with the two remaining hydrogen atoms at positions 2 and 6 in a nuclear hyperfine coupling reaction. The modification of the spin energy of this extra electron by the magnetic f ields of these two hydrogens would be expected to sp l i t the signal into a number of hyperfine structures. The number of structures can be calculated in the following way. 113 In a magnetic f i e ld the Z component of the~magnetic moment of the proton can have either of two possible values + \ and - 4. Since this spin can interact with two hydrogens, in the case of ga l l i c acid, one would assume that there are 4 possible combinations, each of equal intensity. However, since these protons are of an equivalent nature' (positions 2 and 6 are equivalent in ga l l i c acid) the +h, -% and +h situation cannot be distinguished and the result is 3 peaks with an intensity of 1:2:1. This is observed in our measurements of ga l l i c acid and are diagrammat i c a l l y represented in Figures 13, 15 and 16. The hyperfine coupling constants (the distance between peaks measured in gauss) would also be expected to be identical and this was also observed to be the case. The constants measured for ga l l i c acid agree with those published by Dixon and Murphy, 1975. ESR signals were measured for ga l l i c acid under two forms of oxidizing conditions. At high pH the spectra generated were of a very high intensity. This would reflect a very large free radical concentration. Enzymatic oxidation produced an identical signal with identical hyperfine sp l i t t ing constants. However, the intensity was greatly diminished and required maximum gain settings. Increasing the concentration of enzyme only marginally increased the intensity of the s ignal . Enzyme concentrations of greater than 50 ug/ml were found to enhance quinone polymerization with subsequent loss of s ignal. What is significant in these measurements is the fact that peroxidase and tyrosinase, two enzymes which are prevalent in the body, were capable of simulating oxidative conditions and led to free radical formation under simulated biological conditions. Protocatechuic acid, or 3,4-dihydroxybenzoic acid is completely analogous in structure to ga l l i c acid, but is missing a hydroxyl group at position 5. Because of this i t lacks some degree of resonance 114 stabi l izat ion and produces a s l ightly weaker signal. The extra electron generated during oxidation is now able to interact with 3 protons at position 2,5 and 6. We would therefore expect the hyperfine sp l i t t ing to occur as 8 separate peaks. This is i l lustrated in Figure 12. However, since none of the,protons are equivalent in nature, there would be no overlap as was seen in ga l l i c acid and the coupling constants would be expected to di f fer from proton to proton. This is what was observed. The constants measured are in agreement with those published by Kalyanaraman and Sealy, 1982. Caffeic acid or 3,4-dihydroxycinnamic acid is analogous to protocatechuic acid in structure, but possess the cinnamic acid structure instead of the benzoid acid group. Its resonance s tabi l izat ion structure is also similar to protocatechuic acid and i t is therefore not surprising that the signal intensities were also very similar. However, the two additional protons on the cinnamic acid group, increased the complexity of signal considerably. Under ideal conditions 5 protons would result in 32 hyperfine coupling interactions. At high pH with a low time constant we were able to resolve 29 of these peaks (Figure 17). Because of overlap of these peaks, 3 appeared obscured at this level of measurement. The hyperfine coupling constants measured under these conditions are the same as those measured by Dixon et a l . , 1975. When the time constant was increased and some of the resolution lost , 7 major groups of peaks could be observed (Figure 18). These peaks correspond to the peaks resolved for the much weaker enzymatic oxidation state of caffeic acid. Because of the weakness of this s ignal , i t was not possible to resolve out the f u l l 32 hypothetically present. It i s possible to say, however, that peroxidase, tyrosinase and alkaline conditions a l l produced the same parent phenolic free radical (Figure 18, 19 and 20). 115 The Importance of Phenolic Oxidative By-Productsr The importance of such oxidative by-products has been recorded extensively in the l i terature (reviewed by T r o l l , 1982). Hydrogen peroxide, the f i r s t oxidative by-product to be examined in this thesis, has been found to induce genetic damage in a variety of ways. It is an intermediate product in many radical reactions and i t can degrade spontaneously into OH" radicals or continue to react by way of radical intermediates.- In DNA, H^O^ produces base changes, single-strand breaks and inhibits repl icat ion, but produces v i r tua l ly no point mutations (Freese, 1971; Stich et a l . , 1978; Bradley et a l . , 1979). It has been found to be toxic to c e l l cultures (Peterkofsky and Prather, 1977) and has been found to induce chromosome aberrations in both ascites tumours in mice (Schoneich, 1967) and CHO cel ls (Stich et a l . , 1978) . Recently, Speit et a l . , 1982, has shown i t to induce s ister chromatid exchanges in V-79 Chinese hamster ce l l s . Tradit ional Ames test strains are not sensitive to the effects of in solution. This has led to the development of a new series of strains known as the TA102 tester strains. These strains possess A'T base pairs at the site of mutation which are sensitive to oxidative mutagens. They are therefore able to demonstrate considerable genetic damage in the presence of hydrogen peroxide (Levin et a l . , 1982). Carcinogenic potential has been demonstrated by Ito et a l . , 1981. Here, they were able to show a prevalence of gastro-duodenal nodules in mice treated with ^2^2' Because of the low concentrations of 2^^ 2 a t t a l n a b l e in vivo, i t seems dubious that i t s toxicity is due to direct attack. More l ike ly i t s react ivity with another oxidation product, the superoxide or O2' radica l , to form hydroxyl radicals (OH') is responsible for i t s in  vivo effects. 116 Below are l i s ted possible reaction mechanisms for the production of OH* radicals. The reactions outlined do not represent a complete l i s t , but do i l lus trate the interdependence of free radical by-products in the development of DNA damage. e + 0 2 «—•• 0 2 v (1) e~ + 0 2 v + 2H+ <*—• H 2 0 2 (2) e~ + H 2 0 2 + H + « — • HO* + H20 (3) e~ + HO* + H + * — • H 20 (4) from Forman and Boveris, 1982. The OH* radical is the most potent oxidant known. Because of i t s ins tab i l i ty or high react iv i ty , i t is d i f f i cu l t to measure. Some success has been reported using ESR - spin trapping techniques, but the complexity of the spin adduct spectra make this technique a d i f f i cu l t one to pursue. What is more easily measured are the reaction products of hydroxyl radical reactions. Chemical physical measurements have demonstrate that the hydroxyl radical produces single strand breaks in DNA and has also been implicated in the formation of several types of base and nucleoside damage. In the case of strand breakage, i t appears that cleavage occurs on the C,.' or C^' H's with production of a DNA radical leading to phosphodiester bond breaks. Base nucleoside damage is most frequently in the form of OH adducts at the C5 - C6 bond of pyrimidines and imidazole ring breakage in the case of purines (Scholes, 1978). This form of damage can lead to sequence miscoding and local denaturation of the double strands. Superoxide, a free radical found to be present in solutions of phenolic acids, is also a potent oxidant. Its significance in the 117 induction of chromosomal damage appears to be considerable. Three pathways which are of particular interest are outlined in Figure 24. Here i t may be seen that superoxide can: oxidize SH groups to S-S bridged groups (Pathway 1);- dismute to form ^2^2 P ^ u s ground-state oxygen (Pathway 2) or react with ferr ic ions to form ferrous ions (Pathway 3) . Each pathway may lead to significant changes in ce l lu lar metabolism (from Oberley & Buettner, 1979). In Pathway 1, the formation of disulfides can result in conformational changes of essential proteins. This could result in the activation or inactivation of key enzymes required in ce l lu lar metabolism and div is ion. The role of sulfhydryl groups in carcinogenesis has been reviewed by Harington, 1967. Pathway 2 i l lus trates how superoxide can dismute to form ^2^2 and ground-state oxygen. Dismutation occurs much more rapidly in the presence of superoxide dismutase and the consequence of the production of H^O^ has already been discussed. The last pathway, in which superoxide donates electrons to metals may also have serious consequences for ce l lu lar metabolism. By altering the oxidation states of essential transit ion metals, i t is possible to also alter the overal l oxidation -reduction potential of a c e l l . Transformed cel ls appear to be part icularly sensitive to such changes (Fernandez-Pol et a l . , 1977). Emerit et a l , 1982, have recently demonstrated that superoxide radicals are capable of inducing chromosome breakage and sister-chromatic exchange. It is this form of act iv i ty which becomes evidence in the induction of chromosomal abnormalities in CHO ce l l s . Perhaps one of the most outstanding features of the ab i l i t y of superoxide radicals to cause damage, is their ab i l i t y to traverse ce l lu lar barr iers . Superoxide anion radicals have been shown to cross membranes of erythrocytes (Lynch and Fridovich, 1978) and granulocytes 118 Figure 24: Major pathways for the induction of ce l lu lar damage by the superoxide anion free radica l . From Oberley and Buettner, 1979, p. 1144. 119 120 (Gennaro and Romeo, 1979) through anion channels. Superoxide anion radicals have also been shown to cross a r t i f i c i a l l i p i d bilayer membranes at temperatures above the l i p i d phase-transition (Rumyantseva et a l . , 1979). It is therefore possible that intracel lu lar ly generated superoxide can pass direct ly through l i p i d membranes which are f lu id at 37° (Powis et a l . , 1981). This evidence would suggest that although phenolics may be localized in their effects, their generation of superoxide radicals may be less so. Other Dietary Sources of Free Radical Oxidation Products: The a b i l i t y of plant phenolics to generate hydrogen peroxide and free radical oxidation products does not appear to be unique. Several other components of diet have been reported to share this act iv i ty . Vitamin C, or ascorbic acid, is a required cofactor in some oxidation reactions requiring molecular oxygen. It has been reported to cleave DNA and this cleavage has been found to be oxygen dependent (Stich et a l . , 1976). Sodium ascorbate has been shown to increase the frequency of s ister chromatid exchanges (a sensitive indicator of DNA damage) (Speit et a l . , 1980) and has been shown to induce mutations in S. typhimurium (Stich et a l . , 1978). The induction of DNA damage appears to be catalase sensitive (Stich et a l . , 1979; Peterbofsky and Prather, 1976) suggesting that hydrogen peroxide may be the cause of the damage. Superoxide dismutase has also been shown to reduce DNA damage, implicating superoxide radicals as an alternative cytotoxic agent (Morgan et a l . , 1976). In a paper by Yamaguchi, 1981, ascorbic acid, as well as epinephrine, cytochrome c, pyrogallol , pyrocatechol and menadione, were a l l found to be sensitive to these enzymes. Pyrolysates of tryptophan, glutamic acid and globulin were also 121 found to be mutagenic in S. typhimurium strains, "TA98 and TA100 and this act ivi ty could be reduced by the presence of myeloperoxidase (Yamada et a l . , 1979). This would suggest again, that peroxides are significant factors in the mutagenic act iv i ty of amino acid and protein pyrolysates. They also appear to be the active components of autooxidized fatty acids (Yamaguchi et a l . , 1980). Biological Protection Mechanisms: Obviously, organisms have evolved mechanisms to protect themselves against the genotoxic effects of such oxidation by-products. In fact, an early explanation for the different oxygen tolerances of aerobes and obligate anaerobes was based on H^O^ tolerance. Thus, aerobes were thought to contain catalase as a defence mechanism against H^O^, while anaerobes lacked this enzyme and were subsequently k i l l e d by 2^^ 2 w n e n exposed to oxygen levels present in a i r . With the discovery of the more short-lived oxygen free radicals , i t became obvious that other enzymes must exist which act in a protective fashion toward free radical damage. Thus, superoxide dismutase was discovered and extensively characterized by McCord and Fridovich (1969). The evolutionary development of such enzymes can only serve to i l lus trate the importance of oxidative reactions to higher animals. One would expect, then, that susceptibi l i ty to oxidative by-products might depend on the presence of such enzymes to act in a protective role . This appears to be the case. In such tumor c e l l lines as Ehrlich ascites tumor ce l l s and Morris hepatoma ce l l s , reduced levels of superoxide dismutase have been observed (Dionisi et a l . , 1975; Sahu et a l . , 1977). These cel ls are particularly sensitive to the effects of free radical producing drugs such as bleomycin and streptonigrin. In both cases i t was concluded that superoxide radical was one of the 122 mediators for the enhancement of DNA chain breakage (Sausville et a l . , 1978; Cohen et a l . , 1963). Preferential k i l l i n g of these tumor lines is thought to explain their effectiveness. Oxidation Reactions in Man: There seems l i t t l e doubt that the ingestion of phenolics in the diet of man has far-reaching consequences in the carcinogenic process. Oxidation of these phenolics has been shown to lead to polymerization of quinone products with concurrent production of activated oxygen species. Factors which appear to favour the oxidative process include time, increased oxygen-pressure, high pH and the presence of transition metals. One must consider, then, whether such situations arise in vivo. In man, the average transit time for food to pass through the body is about 8 hours (Mariella and Blau, 1968). It might, therefore be expected that a higher percentage of the f inalized oxidation products would be found in the large intestine and colon. Since man is an aerobic organism, the oxygen pressure of blood hemoglobin is about 100 mm Hg in ar ter ia l blood dropping to approximately 35 mm Hg in the veins (Cl in ica l Hematology, 1981a). These levels are well within the ranges used to measure the oxidation of phenolics reported in this thesis. The pH values of various organs in man tend to vary from acidic levels in the stomach of between 1.0 and 3.0 (CRC Handbook, 1982) and the relat ively alkaline areas of the pancreas and duodenum at pH 7.5 -8.6 (Mariella and Blau, 1968). We would therefore expect rates of oxidation to be greatest in areas of highest pH. Levels of transition metals are more d i f f i c u l t to measure. Blood tends to be relat ively high in iron (approximately 18 umol/1 in serum) and copper (13 - 23 umol/1 of 123 whole blood) (Cl in ica l Hematology, 1981b) but these values are largely determined by the health and diet of the individual . We might therefore conclude that the oxidative process of phenolics i s possible in man. There are, however, several mitigating factors. The f i r s t and-primary one is that phenolics may be metabolized in man to glycosides and glucuronate derivatives. The metabolic fate of protocatechuic acid has been documented by Scheline, 1966, who found that i t was -excreted largely unchanged, but partly combined with glucuronic and sulphuric acids in rabbits. DeEds et. a l . , 1957, has shown that i t may also be methylated in rats and rabbits to v a n i l l i c acid (4-hydroxy-3-methoxybenzoic acid). The metabolism of ga l l i c acid after oral or intraperitoneal administration to the rat and rabbit has been studied by Booth et a l . , 1959. This group found that the major urinary metabolite besides ga l l i c acid i t s e l f , was 3,5-dihydroxy-4-methoxybenzoic acid. Intestinal microflora appear to have a marked effect on the metabolism of these compounds. The hydrolysis of glucuronides appears to occur in the presence of these organisms. This may be an important feature in the metabolism of compounds excreted in the b i l e as glucuronide conjugates (Scheline, 1966). There is no information to date, as to whether these metabolically altered phenolics are capable of oxidizing and forming peroxides and free radical metabolites. The body also possesses several enzyme systems for the detoxi f i -cation of activated oxygen species. Catalases, peroxidases and superoxide dismutases have a l l been discussed in some detai l as to their role in protecting organisms from the toxic effects of oxidative by-products. The role of such enzymes cannot be underestimated in the body. As was shown in Table 2, the l iver homogenate system known as S9 was able to reduce the clastogenic act iv i ty of a l l phenolic substances measured. 124 S9 is a term used to refer to the 9000 x g "supernatant fraction of ra t - l i ver homogenate prepared from male rats pretreated with a single 0.5 g/Kg intraperitoneal dose of Aroclor 1254 (a commercial mixture of polychlorinated biphenyls). Although this is largely a chemically undefined mixture, inherent to this particular fraction are the l iver microsomes. The biochemical composition of the microsomal fraction has been extensively characterized and has been found to contain a wide variety of enzymes, including cytochrome oxidases, ' catalases, hydroxylases, and various l y t i c enzymes (Amar-Costesec et a l . , 1974). It is therefore not surprising that S9 was capable of altering the clastogenic act iv i ty of the phenolic substances tested. We are faced therefore, with an overwhelming variety of conditions by which ingested phenolics might play a role in the process of carcinogenesis. Phenolics in the Process of Carcinogenesis: The process of carcinogenesis is believed to occur as a prolonged, multistep development which is usually divided into two stages. The f i r s t stage, is considered to be an in i t i a t ion phase where a low dose of a true carcinogen is applied to a target tissue. This is believed to be followed by a promotion phase where repeated applications of such substances act to enhance the production of a malignant tumour. Evidence for this process is reviewed by Berenblum and Shubik, 1979, for a model system of skin carcinogenesis in mice. Phenolics in the Ini t iat ion of Carcinogenesis: The "init iat ion" of a tumour requires by definit ion an irreversible change in one or more of the ce l l s of the target tissue. This is believed to be the result of a mutation or alteration in the genetic 125 material of the c e l l . As has been discussed previously, the oxidative by-products, 2^^ 2 a n t ^ ^2*' a r e capable of inducing strand breaks and mutations in exposed ce l l s . One might hypothesize, then, that organ specifity might arise in areas which are conducive to the oxidation of phenolics. Safrole, a carcinogenic plant phenolic isolated from the genus Heterotropa, appears to be. an example of this phenomenum. This compound is c lass i f ied as a weak hepatocarcinogen and is metabolized by the l iver to i t s 1'-hydroxy derivative. The 1'-hydroxy metabolite is a stronger hepatocarcinogen than the parent compound (Miller et a l . , 1983; Mi l ler et a l . , 1979). It i s possible that the oxidation of safrole in the l i ver might lend i t s e l f to the production of activated oxygen species and the i n i t i a t i o n of a neoplastic process. Phenolics as Promotors of Carcinogenesis: Recent studies have demonstrated that tumour promotors can damage DNA without reacting with i t d irect ly . One of the best known promotors, TPA (a phorbol ester 12-0-tetradecanoylphorbol-13-acetate) has been found to stimulate cel ls by producing activated forms of oxygen, including superoxide anion radicals and peroxides, agents which are free radicals or generators of free radicals (Marx, 1983). The clastogenic act iv i ty of these free radical products has already been discussed. However, many of the mechanisms of how tumour promotion occurs, remains l i t t l e understood. Free radical-induced chromosomal rearrangement of ce l lular oncogenes might explain how genes enter a "de-regulated" state. If separated from adjacent repressor genes, these oncogenes might find themselves able to be expressed. This would eventually lead to a transformed state. 126 Recent evidence has shown that this may be the case. Birnboim, 1982 has published a paper in which a correlation is found between the tumour-promoting ab i l i t y of a compound and the amount of DNA damage induced. They have found that inhibitors of TPA-induced promotional ac t iv i ty , also reduce the number of DNA strand breaks. Moreover, DNA damage was also found to be prevented by enzymes which block formation of superoxide and peroxide, such as superoxide dismutase and catalase. Inhibitors of these enzymes were found to restore the tumour-promoting activity of the compounds tested. Amplification of these effects may occur in the white blood ce l l s . Polymorphonuclear leukocytes or PMN's are a type of phagocytic immune c e l l . When stimulated, they undergo an "oxidative burst" with marked increases in oxygen consumption as well as production of superoxide radicals and hydrogen peroxide. It is believed that i t is the production of these activated oxygen species which allows them to k i l l and scavenge antigenic material. When PMN's or other phagocytes are treated with such well known promotors as TPA, they are found to undergo an oxidative burst seconds after application of the compound (Goldstein et a l . , 1981). The subsequent production of superoxide and peroxide may therefore act to amplify the promotor's i n i t i a l effect. This observation may explain why cel ls are so sensitive to the application of promotor substances. In the previous discussion, evidence has been presented which confirms that during the process of oxidation, plant phenolics are capable of producing peroxide and free radical intermediates. Under conditions favouring oxidation, then, we might expect phenolics to act as promotors of carcinogenesis. 127 At present, evidence for such a case in humans is only speculative. One example may be found in the correlation between betel nut (Areca  betle) chewing and the incidence of ora l , pharyngeal and esophageal tumours (Ranadive et a l . , 1979). A common practice among betel nut chewers is to mix lime with the betel nut and/or tobacco to form a "quid" which is chewed continuously. The high polyphenolic content of betel nut might then be oxidized at the higher pH levels obtained with the use of lime, and a chain of reactions begun leading to circumstances favourable for the production of a tumour. This hypothesis has been reviewed by Stich and Rosin, 1983. The ab i l i t y of plant phenolics to act as promotors of carcinogenesis may also be modulated by other components of diet. The antioxidant, vitamin E , or ct-tocopherol, has received renewed interest as an essential nutrient for humans. A major function of vitamin E, in animals, appears to be that of an antioxidant, inhibit ing both enzymatic and nonenzymatic l i p i d peroxidation (Lehninger, 1970). Vitamin E and several other antioxidants act by competing for reaction with peroxy free radicals , RO2". In this action, free radicals are removed from the system via formation of tocopherol quinones or dimers. In the presence of an antioxidant, the predominant termination reaction w i l l be that involving the antioxidant, and the termination rate w i l l be largely determined by the structure of the antioxidant (Witting, 1980). The s imilarity of structure (tocopherols and phenols) would suggest that under the proper reaction conditions, plant phenolics might also be able to act as antioxidants. This appears to be the case and may be seen i l lustrated as inhibitors of carcinogenesis. 128 Phenolics as Inhibitors of Carcinogenesis: Any discussion of carcinogenic potential must include recent findings that phenolics can also act in an inhibit ing role. Since carcinogenesis appears to be a multistep process, this can occur in several ways. In addition, phenolics, being relat ively reactive molecules also appear to be able to interact in this process in several ways. The f i r s t and most obvious way is for the phenolics to bind direct ly with the carcinogenic substance. Ferul ic , caffeic , chlorogenic and el lagic acids appear to inhibit the mutagenicity of the reactive species of benzo(a)pyrene. They are believed to do this by direct interaction resulting in the formation of mutagenically inactive complexes (Woods et a l . , 1982). Several phenolics have also been observed to bind one of the metabolites of the carcinogen and mutagen, N-Methyl-N-Nitro-N-Nitrosoquanidine or MNNG. In a study using mutagenicity in Salmonella  typhimurium, Stich and Rosin, 1983, were able to demonstrate that addition of phenolics such as ga l l i c acid, caffeic acid, chlorogenic acid and commercially prepared tannins were able to inhibit mutagenicity of MNNG only when applied concurrently with the carcinogen. Phenolics also appear to interact and thereby interfere with the activation of precarcinogens. Aflatoxin Bl or AFB1 requires activation by microsomal mixed function oxidases to become the reactive AFB1-2,3-oxide which is carcinogenic (Lin et a l . , 1978; Neal and Colley, 1978). HPLC analysis of the metabolites of AFB1 would suggest that phenolics suppressed mutagenicity in Salmonella typhimurium by interfering with i t s metabolic activation (Chan, 1982). 129 Phenolics also appear to inhibit carcinogenesis by acting as traps for nitrosation reactions. They accomplish this by reacting with n i t r i t e to form C-nitroso phenolic compounds (Mirvish, 1981; Walker et a l . , 1982). The reactivity of the phenolics in such a reaction appears to depend on the number of hydroxy groups and their position. Phenolics also react with n i t r i t e to produce quinones and nitrous oxide. This pathway involves free radical production, and may lend i t s e l f to promotional act iv i ty . SUMMARY In summary, this thesis has attempted to provide evidence of how phenolic molecules may play a key role in the process of carcinogenesis. Under oxidative conditions they appear to produce significant levels of hydrogen peroxide, superoxide and parent phenolic free radicals . These oxidative products are a l l capable of inducing damage to DNA and may account for the clastogenic act iv i ty of the compounds tested at pH 7.0. The presence of transit ion metals and high pH appeared to increase the rate of production of these products. Activated oxygen species have been implicated as a mechanism for both the in i t ia t ion and promotion of carcinogenic ac t iv i ty . Phenolics, may therefore play a significant role as both in i t ia tors and promotors of carcinogenic act iv i ty by serving as sources of these activated species. Under less oxidative conditions, however, phenolics appear to act as inhibitors of chemical carcinogenesis. 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OH OH OH Phloroglucinol Pyrogallol From Morrison and Boyd, 1973, p. 787. 143 APPENDIX 2: The molecular s t r u c t u r e of i ) cinnamic acids and O H C H = C H C O O H 8) K=R'=K; p-coumaric A c i d 9) R=OH, R=H; c a f f e i c A c i d 10) R=0CH R'=H, f e r u l i c A c i d 11) R=R'=0CH_; s i n a p i c Acid i i ) benzoic acids C O O H C O O H 1) R=R'=H; p-hydroxybenzoic A c i d 6) R=H: s a l i c y l i c Acid 2) R=0H,R'=H; protocatechuic A c i d 7) R=0H; g e n t i s i c A c i d 3) R=0CH_, R*=H; v a n i l l i c A c i d 4) R=R1=0H; g a l l i c A c i d 5) R=R'=0CH • s y r i n g i c A c i d From Ribereau-Gayon, 1972, p. 82. 144 APPENDIX 3: The molecular s t r u c t u r e s of commonly o c c u r r i n g f l a v o n o i d s . HO. OH Naringenin chalcone v O H HO Flavone A p i g e n i n HO V ^ O H Flavanone Naringenin HO HO O F l a v o n o l Kaempferol HO HO O Anthocyanidin P e l a r g o n i d i n I s o f l a v o n e G e n i s t e i n From Hahlbrook, 1981, p. 426. 145 

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