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Development of a ³²P-postlabeling assay for O⁶-methylguanine Lauener, Ronald William 1988

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DEVELOPMENT OF A P-POSTLABELING ASSAY FOR 0 -METHYLGUANINE By RONALD WILLIAM LAUENER B.S c , Simon Fraser U n i v e r s i t y , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (GENETICS PROGRAM) We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © Ronald W i l l i a m Lauener, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Medical Genetics The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 19, 1988  DE-6H/8-n i i ABSTRACT Mo n i t o r i n g of the promutagenic DNA adduct, O^-methylguanine, i n the e x f o l i a t e d c e l l s (e.g. o r a l mucosal) of t i s s u e s from i n d i v i d u a l s exposed to t o b a c c o - s p e c i f i c N-nitrosamines may a i d i n the e v a l u a t i o n of t i s s u e - s p e c i f i c r i s k of ca r c i n o g e n e s i s . People w i t h h i g h l e v e l s of t h i s adduct c o u l d be i d e n t i f i e d and the appropriate i n t e r v e n t i o n taken. Current techniques f o r d e t e c t i o n of O^-methylguanine are u n s u i t a b l e f o r the measurement of low adduct l e v e l s i n s m a l l t i s s u e samples. 39 This t h e s i s describes the development of a P - p o s t l a b e l i n g method f o r d e t e c t i o n of O^-methylguanine i n microgram amounts of DNA. I n the f i r s t p a r t of the p r o j e c t I synth e s i z e d O^-methyldeoxyguanosine 3'-monophosphate (0^mdG3'p), needed as a chromatography marker. This was achieved u s i n g a two step approach; p r e p a r a t i o n of 0 -methyldeoxyguanosine (0 mdG) fo l l o w e d by chemical p h o s p h o r y l a t i o n w i t h K^PO^ i n formamide. The i d e n t i t y of the compound was confirmed by U.V. spectroscopy and enzymatic a n a l y s i s . This i s the f i r s t r e p o r t e d s y n t h e s i s of the modifi e d n u c l e o t i d e . Using the s y n t h e t i c marker, h i g h performance l i q u i d chromatography (HPLC) procedures were then developed f o r i s o l a t i o n of 0^mdG3'p from d i g e s t e d DNA p r i o r to p o s t l a b e l i n g . ft 39 The b a s i c method f o r a n a l y s i s of 0 -methylguanine by P - p o s t l a b e l i n g comprises f i v e steps, a) d i g e s t i o n of DNA c o n t a i n i n g 0^-methylguanine to deoxynucleoside 3'-monophosphates u s i n g m i c r o c o c c a l nuclease and spleen phosphodiesterase, b) i s o l a t i o n of 0^mdG3'p from normal n u c l e o t i d e s u s i n g reverse-phase HPLC, c) 3 2 P - l a b e l i n g of 06mdG3'p to give 0 6-methyldeoxyguanosine 3',5' 3 2P-bisphosphate (06mdG3'5'p) u s i n g 3 2P-ATP and p o l y n u c l e o t i d e kinase, d) 2-dimensional polyethyleneimine c e l l u l o s e t h i n l a y e r chromatography (PEI-TLC) to r e s o l v e 0^mdG3'5'p from other r a d i o a c t i v e i i i m a t e r i a l s and e) autoradiography and s c i n t i l l a t i o n counting to q u a n t i t a t e 06mdG3'5'p. Several v a r i a t i o n s of the b a s i c technique were then i n v e s t i g a t e d w i t h the goal of improving the s e n s i t i v i t y . One m o d i f i c a t i o n , encompassing a second HPLC p u r i f i c a t i o n of 0^mdG3'5'p showed the g r e a t e s t s e n s i t i v i t y : 0.5 micromole 0^-methylguanine per mole normal n u c l e o t i d e (0.5 jumole/mole) . Using t h i s method, 0^-methylguanine was detected i n the DNA of mammalian t i s s u e - c u l t u r e c e l l s t r e a t e d w i t h an agent, N-methyl-N-nitrosourea (MNU) known through independent techniques to form t h i s adduct. 32 The development of P - p o s t l a b e l i n g methods f o r d e t e c t i o n of other small DNA adducts i s f e a s i b l e u s i n g the approach described here f o r 0^-32 6 methylguanine. The performance of the P - p o s t l a b e l i n g method f o r 0 -methylguanine equals t h a t of the best a v a i l a b l e methods when microgram q u a n t i t i e s of DNA are assayed. This new method w i l l complement e x i s t i n g techniques. i v CONTENTS Page ABSTRACT i i CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS x i GENERAL INTRODUCTION 1 1. Environment and Human Cancer 1 2. An Overview of Chemical Carcinogenesis 1 3. DNA Adducts and Chemical Carcinogenesis 2 4. D e t e c t i o n of DNA Adducts i n Humans-Molecular Cancer Epidemiology 5 32 5. P - P o s t l a b e l i n g Assays to Measure DNA Damage 6 6. The S i g n i f i c a n c e of 0^-methylguanine i n Carcinogenesis 11 6.1 Formation of 0^-methylguanine 11 6.2 H i s t o r i c a l P e rspectives 12 6.3 Repair of 0^-methylguanine 12 6.4 C o r r e l a t i o n of 0^-methylguanine P e r s i s t e n c e w i t h Carcinogenesis by N-Nitrosamines 14 6.5 The Molecular Mechanism of Mutagenesis by 0 -methylguanine 15 6.5.1 I n V i t r o 15 6.5.2 I n Vivo 16 6.6 I n i t i a t i o n of Carcinogenesis through A c t i v a t i o n of C e l l u l a r Proto-oncogenes by 0^-methylguanine Induced Mutation 17 V 7. U l t r a - s e n s i t i v e Methodology f o r D e t e c t i o n of 0 -methylguanine DNA Adducts 18 8. Current Problem 19 9. Obj e c t i v e s 20 MATERIALS 21 METHOD DEVELOPMENT 24 1. Synthesis of O^-methyldeoxyguanosine 3'-monophosphate 24 I n t r o d u c t i o n 24 Methods and Re s u l t s 26 1.1 P r e p a r a t i o n of O^-methyldeoxyguanosine 26 1.2 P r e p a r a t i o n of O^-methyldeoxyguanosine 3'-monophosphate 32 2. 0 -methylguanine Q u a n t i t a t i o n by P - P o s t l a b e l i n g A n a l y s i s 40 I n t r o d u c t i o n 40 Methods and Re s u l t s 43 2.1 Method #1: S i n g l e HPLC P u r i f i c a t i o n - D i p h o s p h a t e L e v e l 43 2.2 Method #2: Dual HPLC P u r i f i c a t i o n - D i p h o s p h a t e L e v e l 53 2.3 Method #3: S i n g l e HPLC Purification-Monophosphate L e v e l 54 2.4 Method #4: Dual HPLC Purification-Monophosphate L e v e l 55 3. Performance of the P - P o s t l a b e l i n g Assays f o r 0 -methylguanine 58 I n t r o d u c t i o n 58 Methods and Re s u l t s 60 3.1 R e p r o d u c i b i l i t y of the Assay f o r Syn t h e t i c 0^mdG3'p 60 3.2 R e p r o d u c i b i l i t y of DNA D i g e s t i o n 60 c 3.3 Recovery of 0 -methyldeoxyguanosine 3'-monophosphate 63 3.4 S e n s i t i v i t y 64 v i DETECTION AND QUANTITATION OF O6-METHYLGUANINE IN THE DNA OF TISSUE-CULTURE CELLS TREATED WITH N-METHYL-N-NITROSOUREA 68 I n t r o d u c t i o n 68 1. D e t e c t i o n and Q u a n t i t a t i o n of O^-methylguanine i n the DNA of CHO C e l l s t r e a t e d w i t h N-methyl-N-nitrosourea 69 Methods 69 Res u l t s 70 2. D e t e c t i o n and Q u a n t i t a t i o n of O^-methylguanine i n the DNA of C3H10T1/2 Mouse Embryo F i b r o b l a s t C e l l s t r e a t e d w i t h N-methyl-N-n i t r o s o u r e a 74 Methods 74 Res u l t s 75 DISCUSSION AND CONCLUSIONS 83 1. Method Development 83 2. Comparison of Methods 84 32 3. Factors L i m i t i n g the Performance of the P - P o s t l a b e l i n g Assays 86 3.1 R e p r o d u c i b i l i t y 86 3.2 Recovery 87 3.3 S e n s i t i v i t y 88 4. D e t e c t i o n and Q u a n t i t a t i o n of O^-methylguanine i n Mammalian Ti s s u e - C u l t u r e C e l l s Treated w i t h N-Methyl-N-nitrosourea 90 5. Recommendations to Enable A p p l i c a t i o n of the Method to Human Studies 90 REFERENCES 93 v i i LIST OF TABLES Page 3 9 Table 1 R e p r o d u c i b i l i t y of the P - p o s t l a b e l i n g assay f o r s y n t h e t i c 0^-methyldeoxyguanosine 3'-monophosphate 61 Table 2 R e p r o d u c i b i l i t y of DNA d i g e s t i o n 62 32 Table 3 S e n s i t i v i t y of the P - p o s t l a b e l i n g assay f o r 0^-methylguanine-method #1 65 32 Table 4 Performance c h a r a c t e r i s t i c s of the P - p o s t l a b e l i n g assays f o r 0 -methylguanine 85 v i i i LIST OF FIGURES Page Figure 1 Schematic r e p r e s e n t a t i o n of the mu l t i s t a g e process of 3 carcinogenesis Figure 2 Bas i c procedure f o r d e t e c t i n g DNA-carcinogen adducts u s i n g 3 2 P - p o s t l a b e l i n g 7 Figure 3 Chemical s t r u c t u r e of 06mdG3'p 25 Figure 4 HPLC e l u t i o n p r o f i l e of isomeric methyldeoxyguanosines r e s u l t i n g from the methylation of deoxyguanosine w i t h e t h e r e a l diazomethane 27 Figure 5 HPLC chromatogram of once p u r i f i e d O^ mdG 29 Figure 6 HPLC a n a l y s i s of twice p u r i f i e d 06mdG 30 Figure 7 U.V. spectrum of 06mdG i n ddH 20 31 Figure 8 HPLC a n a l y s i s of 0 6mdG/formamide/KH 2P0 4 p h o s p h o r y l a t i o n mixture before (A) and a f t e r (B) 1.5 hours h e a t i n g at 100°C 33 Figure 9 P u t a t i v e O^ mdG monophosphates i n crude phosph o r y l a t i o n mixture, separated by semi-preparative HPLC. 34 Figure 10 HPLC a n a l y s i s of isomeric 0^mdG3' and 5'-monophosphates i s o l a t e d from the r e a c t i o n mixture 36 Figure 11 HPLC e l u t i o n p r o f i l e s of p u r i f i e d 06mdG5'p (A) and 06mdG3'p (B) 37 Figure 12 U.V. spectrum of 06mdG3'p i n ddH 20 38 Figure 13 C h a r a c t e r i z a t i o n of 0^mdG3'p us i n g 5'-nucleotidase of C. adamanteus venom 39 Figure 14 Conversion of 06mdG3'p to 06mdG by nuclease PI 41 32 Figure 15 1-Dimensional PEI-TLC autoradiogram of p u t a t i v e P-labeled 0 mdG3'5'p de r i v e d from l a b e l i n g of 0 mdG3'p w i t h gamma P-ATP and p o l y n u c l e o t i d e kinase 42 32 ft Figure 16 Summary of the P - p o s t l a b e l i n g methods f o r 0 -methylguanine 44 Figure 17 I s o l a t i o n of 0^mdG3'p from DNA dig e s t e d w i t h m i c r o c o c c a l nuclease and spleen phosphodiesterase u s i n g reverse-phase HPLC. 47 Figure 18 Representative autoradiograms f o r p o s t l a b e l i n g a n a l y s i s of O^-methylguanine i n 1 /ig DNA us i n g method #1 ( s i n g l e HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) 51 i x 32 Figure 19 Representative autoradiograms f o r P - p o s t l a b e l i n g a n a l y s i s of 0 -methylguanine i n 1 fig DNA u s i n g method #3 ( s i n g l e HPLC purification-monophosphate l e v e l ) 56 32 fi Figure 20 P u r i f i c a t i o n of P-labeled 0 mdG5'p us i n g reverse-phase HPLC (method #4) 57 o n c Figure 21 Sample autoradiograms f o r P - p o s t l a b e l i n g a n a l y s i s of 0 -methylguanine i n 1 fig DNA u s i n g method #4 (dual HPLC purification-monophosphate l e v e l ) 59 32 6 Figure 22 S e n s i t i v i t y of the P - p o s t l a b e l i n g assay f o r 0 -methylguanine-method #1 ( s i n g l e HPLC p u r i f i c a t i o n -diphosphate l e v e l ) 66 32 6 Figure 23 S e n s i t i v i t y of the P - p o s t l a b e l i n g assay f o r 0 -methylguanine-method #2 (dual HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) 67 Figure 24 D e t e c t i o n and q u a n t i t a t i o n of 0^-methylguanine i n CHO c e l l s t r e a t e d w i t h MNU 72 Figure 25 Dose-response of 0^-methylguanine i n the DNA of CHO c e l l s t r e a t e d w i t h MNU 73 Figure 26 D e t e c t i o n and q u a n t i t a t i o n of 0^-methylguanine i n the DNA of C3H10T1/2 c e l l s t r e a t e d w i t h MNU (method #1) 77 Figure 27 Autoradiograms of DNA from C3H10T1/2 c e l l s exposed to v a r i o u s doses of MNU (method #2) 79 Figure 28 Dose-response of 0 6-methylguanine i n the DNA of C3H10T1/2 mouse-embryo f i b r o b l a s t c e l l s t r e a t e d w i t h MNU 81 Figure 29 S u r v i v a l of C3H10T1/2 mouse-embryo f i b r o b l a s t c e l l s exposed to MNU 82 LIST OF ABBREVIATIONS 06mdG 0^-methyldeoxyguanosine 06mdG3'p 0^-methyldeoxyguanosine 3'-monophosphate 06mdG5'p 0^-methyldeoxyguanosine 5'-monophosphate 06mdG3'5'p 6 32 0 -methyldeoxyguanosine 3',5' P-bisphosphat< dA3'p deoxyadenosine 3'-monophosphate dA3'5'p 32 deoxyadenosine 3'5' P-bisphosphate MNU N-methyl-N-nitrosourea AT 0^-alkylguanine-DNA-alkyltransferase HPLC hi g h performance l i q u i d chromatography PEI-TLC polyethylene-imine t h i n l a y e r chromatography fimole micromole (10"^ mole) Mg microgram (10"^ gram) Ml m i c r o l i t e r ( 10~ 6 l i t e r ) x i ACKNOWLEDGEMENTS I wish to express my s i n c e r e a p p r e c i a t i o n : to Dr. Bruce Dunn (Environmental Carcinogenesis U n i t ) f o r h i s i n v a l u a b l e a i d i n d e v i s i n g an i n t e r e s t i n g and c h a l l e n g i n g p r o j e c t , f o r p r a c t i c a l a s s i s t a n c e and many h e l p f u l d i s c u s s i o n s , and f o r c r i t i c a l readings of t h i s manuscript; to my s u p e r v i s o r , Dr. H.F. S t i c h (Dept. of Zoology and E n v i r o n m e n t a l Carcinogenesis U n i t ) and members of my ad v i s o r y committee, Dr. R.H.C. San (Dept. of Medical Genetics and Environmental Carcinogenesis U n i t ) and Dr. C.J. Eaves (Dept. of Medical Genetics and Terry Fox Laboratory) f o r t h e i r c o n t i n u a l guidance and counsel throughout my p r o j e c t ; to Mr. Dan Twa f o r h i s e x c e l l e n t t e c h n i c a l a s s i s t a n c e and sense of humor; to the s t a f f and students of the Environmental Carcinogenesis U n i t and B.C. Cancer Research Centre f o r p r o v i d i n g a s t i m u l a t i n g s c i e n t i f i c atmosphere i n which to study; and f i n a l l y to my f a m i l y and f r i e n d s f o r t h e i r support and encouragement throughout t h i s work. 1 GENERAL INTRODUCTION 1. Environment and Human Cancer The m a j o r i t y of human cancers are induced by exogenous agents i n the environment. This c o n c l u s i o n a r i s e s p r i m a r i l y from e p i d e m i o l o g i c a l data ( d e s c r i p t i v e and a n a l y t i c a l ) i n d i c a t i n g temporal trends i n incidence and changing r i s k s i n migrant populations ( D o l l et a l . 1966, Waterhouse et a l . 1982, K o l o n e l et a l . 1980). Genetic and c o n g e n i t a l f a c t o r s are b e l i e v e d to be re s p o n s i b l e f o r no more than 2% of the t o t a l cancer burden (Higginson and Muir 1979, Knudson 1977). Exposure to chemicals through d i e t , use of tobacco, and occupation may account f o r up to 85% of a l l cancer m o r t a l i t y i n North America ( D o l l and Peto 1981). 2. An Overview of Chemical Carcinogenesis Most of our knowledge concerning the mechanisms of chemical car c i n o g e n e s i s i s n e c e s s a r i l y d e r i v e d from s t u d i e s on cancer i n d u c t i o n or i t s modulation i n experimental animal systems. Many i d e n t i f i e d human carcinogens cause tumors i n animals (Searle 1984). Several carcinogens a c t i v e i n animals were i n i t i a l l y d i scovered because of i n d u s t r i a l exposure and carcinogenesis i n humans; benzo(a)pyrene and ^-naphthylamine f o r example ( D o l l and Peto 1981). There i s a l s o evidence that at the c e l l u l a r l e v e l , humans by and la r g e do not d i f f e r q u a l i t a t i v e l y from experimental animals i n response to carcinogens. Although tumors were c h e m i c a l l y induced i n animals p r i o r to 1940 (Yamagiwa and Ichakawa 1915, Sasaki and Yoshida 1935), i t was not u n t i l 1961 that the covalent b i n d i n g of a carcinogen, dibenz(a,h)anthracene, to the DNA of mouse s k i n was reported (Heidelberger and Davenport 1961). A multitude of ensuing work showed th a t most animal carcinogens b i n d to the bases of DNA to form covalent DNA-carcinogen a d d i t i o n products or adducts (Hemminki 1983). 2 Moreover, the organotropy of the carcinogen c o u l d o f t e n be p r e d i c t e d by the extent of DNA b i n d i n g i n the t a r g e t t i s s u e ( K r i e k et a l . 1984). These studies were p r i m a r i l y conducted u s i n g r a d i o a c t i v e carcinogens, f o l l o w e d by i s o l a t i o n of DNA and i d e n t i f i c a t i o n of the r a d i o a c t i v e adducted bases by chromatography ( B a i r d 1979). Subsequent work i n d i c a t e d t h a t the DNA b i n d i n g of a chemical may be a more accurate p r e d i c t o r of c a r c i n o g e n i c i t y than mutagenicity. Studies f o l l o w i n g the metabolism of carcinogens showed t h a t they e i t h e r d i r e c t l y formed h i g h l y r e a c t i v e e l e c t r o p h i l i c intermediates or are e n z y m a t i c a l l y converted to these species by d e t o x i f i c a t i o n processes. The e l e c t r o p h i l e s or pre-carcinogens are thought to r e a c t w i t h n u c l e o p h i l i c bases of DNA, forming adducts ( M i l l e r and M i l l e r 1981). Recently i t has been shown th a t the DNA adducts formed i n rodents by major c l a s s e s of carcinogens are a l s o induced i n human c e l l s by the same agents (Autrup and H a r r i s 1983). 3. DNA Adducts and Chemical Carcinogenesis A s i m p l i f i e d schematic diagram of the cur r e n t model f o r chemical ca r c i n o g e n e s i s i s represented i n Figure 1 ( H a r r i s 1985). The m u l t i s t a g e process i s d i v i d e d i n t o s e v e r a l steps necessary f o r tumor i n d u c t i o n but which may be separated both temporally, and m e c h a n i s t i c a l l y (Bertram e t a l . 1987). The concepts of tumor i n i t i a t i o n , promotion, conversion and p r o g r e s s i o n have a r i s e n from s t u d i e s i n experimental animal c a r c i n o g e n e s i s . This model may be used as an i n t e l l e c t u a l framework to consider the r o l e that DNA adducts may p l a y i n m u l t i - s t a g e c a r c i n o g e n e s i s . The e a r l i e s t event i n chemical c a r c i n o g e n e s i s , tumor i n i t i a t i o n , i s comprised of s e v e r a l steps, a) exposure to the carcinogen, b) t r a n s p o r t of the carcinogen to the t a r g e t c e l l , c) a c t i v a t i o n to i t s u l t i m a t e e l e c t o p h i l i c m e t a b o l i t e i f the agent i s a pre-carcinogen and d) DNA damage l e a d i n g to an 3 MULTISTAGE CARCINOGENESIS Exposure Initiation Promotion Conversion Progression LATENCY PERIOD * M ^20 x 10* Minutes ^1 -\> 12.775 Days Figure 1 Schematic r e p r e s e n t a t i o n of the multistage process of carc i n o g e n e s i s . 4 i n h e r i t e d change and the p r e n e o p l a s t i c " i n i t i a t e d " c e l l . M e tabolic a c t i v a t i o n and DNA damage probably occurs w i t h i n hours of exposure. Current evidence suggests that DNA r e p l i c a t i o n must occur before r e p a i r of the DNA adduct to create a s t a b l e b i o l o g i c a l l e s i o n . Thus, r e p l i c a t i o n before r e p a i r or e r r o r -prone r e p a i r can f i x the l e s i o n i n DNA as a mutation. The requirement f o r r e p l i c a t i o n may i n p a r t e x p l a i n the h i g h frequency of neoplasms i n p r o l i f e r a t i n g t i s s u e s (Cayama et a l . 1978). Cancer i s thought to be the r e s u l t of complex i n t e r a c t i o n s between m u l t i p l e environmental f a c t o r s and both acquired and i n h e r i t e d host f a c t o r s ( H a r r i s 1983). As such, DNA adducts should only be considered as necessary f o r the i n i t i a t i o n of chemical carcinogenesis but not s u f f i c i e n t f o r tumor i n d u c t i o n . C r u c i a l are determinants of tumor promotion, p r o g r e s s i o n , conversion and f a c t o r s which can enhance or i n h i b i t these processes. The suggestion t h a t p e r s i s t e n t DNA adducts l e a d to i n i t i a t i o n of car c i n o g e n e s i s i s corroborated by two l i n e s of evidence. F i r s t , people who s u f f e r from DNA r e p a i r d e f i c i e n c y syndromes are prone to the development of cancer (Knudson 1977). Examples are xeroderma pigmentosum, Fanconi's anemia and a t a x i a t e l a n g i e c t a s i a . C u l t u r e d c e l l s from these i n d i v i d u a l s are unable to remove DNA m o d i f i c a t i o n s and are h y p e r s e n s i t i v e to the i n d u c t i o n of mutation a f t e r chemical treatment. Second, q u a l i t a t i v e l y s i m i l a r adducts are formed i n animal species both s e n s i t i v e and r e s i s t a n t to the tumorigenic e f f e c t s of a chemical, but o f t e n a greater t o t a l amount of adducts are formed i n t a r g e t organs of s e n s i t i v e species (reviewed by Wogan and G o r e l i c k 1985). 5 4. D e t e c t i o n of DNA Adducts i n Humans- Molecular Cancer Epidemiology Data from animal s t u d i e s and human c e l l s i n v i t r o i n d i c a t e that formation and p e r s i s t e n c e of DNA adducts should be a necessary event i n the i n i t i a t i o n of chemical carcinogenesis i n humans. De t e c t i o n of DNA adducts i n humans could provide i n f o r m a t i o n on the mechanistic l i n k between exposure to cancer causing chemicals and subsequent r i s k f o r tumor development (Maugh 1984). DNA adducts may be a b e t t e r i n d i c a t i o n of r i s k f o r cancer than q u a n t i t a t i o n of exposure because they are markers o f d i r e c t genetic damage. Molecular cancer epidemiology e n t a i l s the establishment of a caus a l r e l a t i o n s h i p between the formation and p e r s i s t e n c e of a p a r t i c u l a r adduct(s) and a p a r t i c u l a r tumor (Perera 1987). I d e n t i f i c a t i o n of an exposure-related DNA adduct i n t i s s u e s at r i s k could a i d i n the t r a c i n g of the chemical r e s p o n s i b l e . D e t e c t i o n of an environmentally r e l a t e d DNA adduct i n d i c a t e s t h a t exposure to genotoxic chemicals has occurred, and t h a t the person i s at r i s k . The value of a DNA adduct as an accurate e a r l y i n d i c a t o r of n e o p l a s i a can be e s t a b l i s h e d only through r i s k v e r i f i c a t i o n by f o l l o w i n g populations w i t h d i f f e r e n t l e v e l s of adducts f o r many years. Once a cau s a t i v e l e s i o n has been i d e n t i f i e d , d e t e c t i o n i n exposed i n d i v i d u a l s may serve to i d e n t i f y increased r i s k and a l l o w o p p o r t u n i t y f o r e a r l y i n t e r v e n t i o n . The d e t e c t i o n of low l e v e l s of DNA damage i n humans has been p o s s i b l e r e c e n t l y due to the development of s e v e r a l u l t r a - s e n s i t i v e methodologies. The 32 most important are P - p o s t l a b e l i n g (reviewed by Watson 1987), immunoassays (reviewed by S t r i c k l a n d and Boyle 1984), and synchronous fluorescence spectrophotometry (Vahakangas et a l . 1985). Each method has p a r t i c u l a r advantages and optimal a p p l i c a t i o n s . I d e a l l y the use of two or more 6 techniques i n a v a l i d a t i o n scheme would serve to o f f s e t problems w i t h s p e c i f i c i t y and accuracy a s s o c i a t e d w i t h use of a s i n g l e method. A v a i l a b i l i t y of these techniques has i n i t i a t e d an e x p l o s i o n i n research concerning the measurement of DNA adducts i n humans at r i s k f o r cancer due to occupation or l i f e s t y l e . Exposure r e l a t e d adducts have been detected i n p e r i p h e r a l blood lymphocytes of foundry workers and i n the placentae and lungs of smokers, f o r example ( H a r r i s et a l . 1985, Everson et a l . 1986). One i n t e r e s t i n g o b s e r v a t i o n i s the d e t e c t i o n of DNA damage i n s o - c a l l e d unexposed populations i l l u s t r a t i n g perhaps the u n a v o i d a b i l i t y of exposure to genotoxic agents i n the environment ( H a r r i s et a l . 1985, Everson et a l . 1986). 32 5. P - P o s t l a b e l i n g Assays to Measure DNA Damage To address the need f o r a s e n s i t i v e and g e n e r a l l y a p p l i c a b l e t e s t to detect d i r e c t l y the presence of c h e m i c a l l y a l t e r e d bases i n DNA, the p o s t l a b e l i n g method was developed by Randerath and co-workers i n 1981 (Randerath et a l . 1981). This t e s t evolved from methods used to analyse modif i e d c o n s t i t u e n t s i n RNA and normal bases i n DNA (Randerath et a l . 1972, Reddy et a l . 1981). The b a s i c procedure encompasses the f o l l o w i n g steps: a) i s o l a t i o n of DNA from c e l l s exposed to carcinogens, b) DNA d i g e s t i o n to a mixture of normal and adducted deoxynucleoside 3'-monophosphates u s i n g m i c r o c o c c a l endonuclease and 32 32 spleen phosphodiesterase, c) t r a n s f e r of P - l a b e l from P-ATP to the 5' p o s i t i o n of d i g e s t i o n products mediated by T4 p o l y n u c l e o t i d e kinase and d) 32 mapping of the P-labeled adduct n u c l e o t i d e s by m u l t i - d i r e c t i o n a l chromatography on P E I - c e l l u l o s e t h i n l a y e r s , f o l l o w e d by autoradiography. A schematic diagram i l l u s t r a t i n g the method i s shown i n Figure 2. The method i s q u a n t i t a t i v e because the extent of DNA adduction can be determined by 7 Carcinogen • adducted DNA Micrococcal endonuclease + spleen exonuclease Ap + Gp + Tp + Cp + m 5 Cp + Xp + Yp + . . | 3 2 P ) phosphate transfer: |>- 3 2 P) A T P + T4 polynucleotide kinase pAp + pGp + pTp + pCp + pm^Cp + pXp + pYp + Removal of normal nucleotides: PEI-cellulose or reversed phase T L C or reversed phase HPLC pXp + pYp + . Separation and detection of adducts: (i) PEI cellulose T L C (ii) autoradiography Maps of ° P-labeled carcinogen-DIMA adducts 32 Figure 2 Basic procedure f or detecting DNA-carcinogen adducts using P-postl a b e l i n g . 8 32 s c i n t i l l a t i o n counting of P-labeled n u c l e o t i d e s on the chromatograms (Randerath e t a l . 1981). In recent years, the o r i g i n a l method has been modi f i e d to accomodate the d e t e c t i o n of a d i v e r s e a r r a y of aromatic and non-aromatic DNA adducts (Gupta et a l . 1982, Reddy et a l . 1984). The chemical c l a s s of adducts detected depends on the chromatographic c o n d i t i o n s employed i n the f i n a l PEI-TLC step. R e s o l u t i o n of bu l k y aromatic adduct deoxynucleoside 3',5' P-bisphosphates r e q u i r e s the use of solve n t s c o n t a i n i n g h i g h concentrations of urea. Smaller, l e s s hydrophobic adducts (e.g. a l k y l adducts) migrate w i t h the s o l v e n t f r o n t under these c o n d i t i o n s and are not r e t a i n e d on the chromatogram. Bulky, aromatic adducts are detected w i t h the gr e a t e s t s e n s i t i v i t y using 32 the P - p o s t l a b e l i n g method. This i s because these compounds can be e f f e c t i v e l y i s o l a t e d as a c l a s s from the normal n u c l e o t i d e s u s i n g chromatography. This i s accomplished by e l u t i o n of l a b e l e d normal n u c l e o t i d e s o f f the chromatograms onto a paper wick u s i n g urea s o l v e n t s , l e a v i n g the aromatic adducts a t the o r i g i n (Gupta et a l . 1982). The extent of DNA damage i n humans exposed to environmental carcinogens 7 8 i s expected to be l e s s than 1 a l t e r e d base i n 10 to 10 normal bases (Everson et a l . 1986, Randerath et a l . 1986). To broaden the a p p l i c a t i o n of the p o s t l a b e l i n g assay to humaa s t u d i e s , the s e n s i t i v i t y of the method has been improved. This has been achieved f o r d e t e c t i o n of aromatic adducts through the e f f i c i e n t removal of normal n u c l e o t i d e s p r i o r to p o s t l a b e l i n g . Pre-32 l a b e l i n g i s o l a t i o n of adducts allows the a n a l y s i s of more DNA u s i n g l e s s P-ATP, r e s u l t i n g i n lower background counts. E x t r a c t i o n of aromatic adducts from the DNA d i g e s t u s i n g n-butanol f o l l o w e d by p o s t l a b e l i n g a f t e r s olvent removal i s one example (Gupta 1985). In another m o d i f i c a t i o n of the procedure, DNA d i g e s t s are i n j e c t e d onto a reverse-phase HPLC column and the normal n u c l e o t i d e s removed by e l u t i o n w i t h ammonium formate s o l v e n t . The 9 aromatic adducts ( r e t a i n e d on the column) are then recovered by e l u t i o n w i t h methanol (Dunn and S t i c h 1986, Dunn et a l . 1987, Dunn and San 1988). The r e s i s t a n c e of n u c l e o t i d e s adducted w i t h aromatic or bulky non-aromatic groups to the 3'-dephosphorylating a c t i o n of nuclease PI i s the b a s i s f o r yet another recent advance (Reddy and Randerath 1986). I n another approach, a 10-100 f o l d increase i n s e n s i t i v i t y was noted when DNA d i g e s t s were l a b e l e d w i t h a l i m i t i n g amount of c a r r i e r - f r e e (high s p e c i f i c a c t i v i t y ) ATP. Adducted n u c l e o t i d e s were l a b e l e d p r e f e r e n t i a l l y over normal n u c l e o t i d e s under these c o n d i t i o n s (Randerath et a l . 1985). Evidence f o r the i d e n t i t y of an adduct detected by the p o s t l a b e l i n g assay may be obtained by co-chromatography w i t h an a u t h e n t i c marker compound. P o s t l a b e l i n g assays f o r the d e t e c t i o n of s p e c i f i c s m all adducts have been developed. Techniques f o r the d e t e c t i o n of 5-methyIcytosine, bromodeoxyuridine, a formaldehyde induced adduct, and N^-(2-oxoethyl)guanine (the p r i n c i p a l v i n y l - c h l o r i d e adduct), have r e c e n t l y been developed (Wilson et a l . 1986, B o d e l l and Rasmussen 1984, F e n n e l l et a l . 1987, Watson et a l . 1987). 5.1 A p p l i c a t i o n s of the P o s t l a b e l i n g Technique The a b i l i t y of numerous chemicals of d i v e r s e s t r u c t u r e to b i n d to DNA i n v i t r o has been reporte d u s i n g the p o s t l a b e l i n g assay (Randerath et a l . 1981, Gupta et a l . 1982). DNA adducts i n b a c t e r i a , and i n mammalian t i s s u e - c u l t u r e c e l l s exposed to p o l y c y c l i c aromatic hydrocarbons have been assayed (Arce et a l . 1987, Dunn and San 1988). The assay has been used e x t e n s i v e l y to measure the DNA adducts formed i n v a r i o u s t i s s u e s of animals exposed to known carcinogens (Lu et a l . 1986). The a n a l y s i s of DNA adducts formed i n t i s s u e s of animals exposed to mixtures c o n t a i n i n g unknown carcinogens (e.g. c i g a r e t t e smoke condensate, d i e s e l exhaust) has been reported (Randerath et a l . 1986, Wong et a l . 1986). P o s t l a b e l i n g a n a l y s i s was used to detect age r e l a t e d 10 adducts i n non-exposed l a b o r a t o r y r a t s (Randerath et a l . 1986). The t i s s u e s of bottom d w e l l i n g f i s h from p o l l u t e d waters show adducts when analysed (Dunn et a l . 1987). C u r r e n t l y , the method i s being e x t e n s i v e l y a p p l i e d to the d e t e c t i o n and q u a n t i t a t i o n of adducts i n humans v o l u n t a r i l y or i n v o l u n t a r i l y exposed to DNA damaging agents. Exposure r e l a t e d aromatic/bulky adducts have been detected i n the placentae, o r a l mucosa, bronchus, white blood c e l l s , b r o n c h i a l t i s s u e and lungs of smokers (Everson et a l . 1986, Everson et a l . 1987, Everson et a l . 1987, Chacko and Gupta 1987, Randerath et a l . 1987). Adducts have a l s o been detected i n the p e r i p h e r a l blood lymphocytes of foundry workers subjected to a known exposure to p o l y c y c l i c aromatic hydrocarbons ( P h i l l i p s et a l . 1987). 5.2 Advantages of J -P-Postlabeling Methods f o r the D e t e c t i o n of DNA Adducts. Sev e r a l f e a t u r e s of the method make i t s u i t a b l e f o r the monitoring of human exposure to genotoxic chemicals and as a t o o l f o r s t u d i e s of chemical carc i n o g e n e s i s (reviewed by Watson 1987). Using t h i s technique, the DNA damaging a b i l i t y of any chemical or mixture i n v i v o or i n v i t r o can be measured. The range of chemicals which can be t e s t e d i s not l i m i t e d by t h e i r a v a i l a b i l i t y i n r a d i o l a b e l e d form. The d e t e c t i o n of DNA b i n d i n g u s i n g t h i s assay does not r e q u i r e p r i o r knowledge of the chemical i d e n t i t y of the adducts or carcinogens. Only 1-10 y,g of DNA i s r e q u i r e d f o r an a n a l y s i s . This makes p o s s i b l e the d e t e c t i o n of DNA adducts i n small biopsy specimens or e x f o l i a t e d c e l l s (e.g. o r a l mucosal) from exposed i n d i v i d u a l s without p o o l i n g the samples. The s e n s i t i v i t y of d e t e c t i o n f o r aromatic/bulky adducts i n a 10 /zg DNA sample i s about 1 adduct i n 1 0 ^ n u c l e o t i d e s or 1-10 adducts per d i p l o i d mammalian genome (Reddy and Randerath 1986). For most adducts the method gives accurate q u a n t i t a t i o n and gives s a t i s f a c t o r y r e p r o d u c i b i l i t y and recovery ( F e n n e l l et a l . 1986, Gupta et a l . 1982). The method i s v e r s a t i l e i n 11 th a t i t allows f o r the simultaneous a n a l y s i s of many d i f f e r e n t DNA adducts i n a s i n g l e DNA sample. Studies employing dozens of t e s t compounds have shown th a t each p a r t i c u l a r chemical or mixture produces a unique f i n g e r p r i n t p a t t e r n of DNA adducts (Reddy et a l . 1984). In p r i n c i p l e the i d e n t i t y of chemical exposure can be i n f e r r e d by comparison of f i n g e r p r i n t s between the t e s t DNA sample and those produced by exposure of DNA to known chemicals. The c r e a t i o n of a databank composed of p o s t l a b e l i n g f i n g e r p r i n t s f o r a l l suspect chemicals has been proposed (Watson 1987) . The assay i s more s p e c i f i c f o r known adducts than immunoassays because a n t i b o d i e s e x h i b i t c r o s s - r e a c t i v i t y w i t h adducts of s i m i l a r s t r u c t u r e ( S t r i c k l a n d and Boyle 1984). The study of adduct b i n d i n g to de f i n e d DNA sequences and genes i s encouraged due to the minimal amount of DNA r e q u i r e d f o r a n a l y s i s (Gupta 1984, Gupta et a l . 1985). F i n a l l y , the method i s p o t e n t i a l l y u s e f u l f o r i n v e s t i g a t i n g the r e p a i r and p e r s i s t e n c e of DNA adducts. I t should a l s o be a p p l i c a b l e to the i n v e s t i g a t i o n of the e f f e c t s of chemopreventative agents and/or metabolic i n h i b i t o r s on adduct fo r m a t i o n / p e r s i s t e n c e . 6. The S i g n i f i c a n c e of 0^-methylguanine i n Carcinogenesis 6.1 Formation of 0^-methylguanine 0^-methylguanine i s formed by the r e a c t i o n of an e l e c t r o p h i l i c diazonium i o n w i t h the 0^ oxygen of guanine i n DNA i n v i t r o or i n v i v o . M e t h y l a t i n g agents (e.g. some N-nitrosamines and N-nitrosamides) r e a c t w i t h DNA g i v i n g t h i s adduct p l u s more than 10 other methyl adducts (Singer 1985). 0^-methylguanine i s a minor product of methylation and u s u a l l y accounts f o r between 0.1% and 7% of a l l methyl adducts. 12 6.2 H i s t o r i c a l P e r s p e c t i v e s c Loveless was the f i r s t to propose t h a t a l k y l a t i o n a t the 0 p o s i t i o n of guanine i s the c r i t i c a l event i n carcinogenesis and mutagenesis by a l k y l a t i n g agents (Loveless 1969). He c o r r e l a t e d the a b i l i t y of chemicals to e f f e c t 0 -me t h y l a t i o n of deoxyguanosine w i t h t h e i r mutagenic a c t i v i t y i n T-even bacteriophages. The cau s a t i v e mechanism, according to Loveless was m i s p a i r i n g of O^-methylguanine w i t h thymine due to deprotonation of guanine n i t r o g e n N"*", l e a d i n g to G/C -»• A/T t r a n s i t i o n s on r e p l i c a t i o n . 6.3 Repair of 0 -methvlguanine The b i o l o g i c a l importance of a l k y l a t i o n a t the 0 p o s i t i o n of guanine i s emphasized by the f a c t t h a t most c e l l s possess a s p e c i a l r e p a i r pathway s o l e l y f o r t h i s damage (Yarosh 1985). Olsson and L i n d a h l were the f i r s t to deduce the nature of t h i s process i n E. coli (Olsson and L i n d a h l 1980). They c observed methyl group t r a n s f e r from the 0 p o s i t i o n of guanine i n DNA to a c y s t e i n e group i n an acceptor p r o t e i n , generating a S-methyl-L-cysteine res i d u e and r e s t o r i n g the guanine base. L a t e r , t h i s a c t i v i t y was a t t r i b u t e d to a 37,000 MWt. p r o t e i n deemed to be O^-alkylguanine-DNA-alkyltransferase (AT) (Demple et a l . 1982). This p r o t e i n i s able to demethylate l a r g e r a l k y l groups, a l b e i t much l e s s e f f i c i e n t l y (Morimoto et a l . 1985). Normally, b a c t e r i a c o n t a i n about 13-60 molecules of AT per c e l l ( M i t r a et a l . 1982). However, on exposure to low l e v e l s of O^-methylguanine producing chemicals, the t r a n s f e r a s e gene i s derepressed and upwards of 3000 molecules per c e l l are produced. This s o - c a l l e d adaptive response i n b a c t e r i a , r e s u l t i n g i n e l e v a t e d l e v e l s of AT, confers r e s i s t a n c e to the mutagenic and t o x i c e f f e c t s of methylating agents (Samson and Cairns 1977). 13 Mammalian ( i n c l u d i n g human) c e l l s have an eq u i v a l e n t enzyme (Renard and V e r l y 1980). There has been some suggestion of a s i m i l a r though l e s s profound adaptive response to low l e v e l s of chemicals i n animal c e l l s (Montesano et a l . 1979). In normal human t i s s u e s and c e l l s the AT a c t i v i t y v a r i e s up to 1,000 f o l d between t i s s u e s and i n d i v i d u a l s (Grafstrom e t a l . 1984). I n a d d i t i o n , the AT a c t i v i t y of human t i s s u e s i s u s u a l l y s e v e r a l orders of magnitude grea t e r than t h a t of the eq u i v a l e n t rodent t i s s u e s ( H a l l et a l . 1985). I n t e r e s t i n g l y , the a c t i v i t y of the enzyme i s h i g h e s t i n t i s s u e s ( l i v e r , d i g e s t i v e t r a c t ) which might be f i r s t exposed to d i e t a r y a l k y l a t i n g agents (Kyrtopoulos e t a l . 1984). Several human tumor c e l l l i n e s and c e l l s from i n d i v i d u a l s w i t h genetic DNA r e p a i r d e f i c i e n c y diseases l a c k the a b i l i t y to r e p a i r O^-methylguanine (Day e t a l . 1980). Consequently, they are h y p e r s e n s i t i v e to the mutagenic, t o x i c and c l a s t o g e n i c e f f e c t s of methylating agents (Domoradziki e t a l . 1984). Mammalian c e l l s l a c k i n g AT a c t i v i t y have been t r a n s f e c t e d w i t h the cloned AT gene (ada) of E. coli, r e s u l t i n g i n the r e s t o r a t i o n of r e p a i r a c t i v i t y (Samson et a l . 1986, J e l i n e k e t a l . 1988). The a c t i v i t y of AT can be diminished by the a d m i n i s t r a t i o n of the f r e e base, O^-methylguanine, to c e l l s (Dolan et a l . 1985). The r e p a i r of O^-methylguanine i n most c e l l s i s b i p h a s i c , c h a r a c t e r i z e d by a r a p i d e a r l y phase f o l l o w e d by slow l a t e r e p a i r ( S h i l o h and Becker 1981). This has been e x p l a i n e d by the greater r a t e of r e p a i r of O^-methylguanine at h i g h adduct l e v e l s ( S c i c c h i t a n o and Pegg 1982). AT has been r e f e r r e d to as a s u i c i d a l enzyme because once i t has reacted w i t h O^-methylguanine only RNA and p r o t e i n s y n t h e s i s can r e s t o r e i t s l e v e l (Yarosh 1985). The enzyme i s not regenerated a f t e r methyl group acceptance and th e r e f o r e i s a dead-end complex. c Thus, the system i s s a t u r a b l e . The l o s s of 0 -methylguanine from DNA i s coupled w i t h s t o i c h i o m e t r i c formation of S-methyl-cysteine and th e r e f o r e i s 14 the e x c l u s i v e mechanism f o r r e p a i r . The t r a n s f e r and accepting of the methyl group are performed by the same p r o t e i n . 6.4 C o r r e l a t i o n of 0^-methylguanine P e r s i s t e n c e w i t h Carcinogenesis by N- Nitrosamines Numerous i n v e s t i g a t o r s have attempted to define a connection between the frequency of tumorigenesis i n a t a r g e t t i s s u e and the p e r s i s t e n c e or l a c k of r e p a i r of 0^-methylguanine. In e a r l y experiments, a f t e r a s i n g l e a d m i n i s t r a t i o n of methylnitrosourea (MNU) to r a t s , 0 -methylguanine was observed to be removed f a r l e s s r a p i d l y from the DNA of the b r a i n (the p r i n c i p a l t a r g e t organ) than the l i v e r which i s not s u s c e p t i b l e to MNU (Kleihues and Margison 1974). I n a s i m i l a r s i n g l e dose t r i a l the long-term f a t e of MNU d e r i v e d 0^-methylguanine was monitored. I n b r a i n , u n l i k e lung, kidney or l i v e r , t h i s m o d i f i e d base tended to p e r s i s t f o r much of the animal's l i f e t i m e (Kleihues and Bucheler 1977). Experimental tumorigenesis by repeated chronic exposure to a low dose of carcinogen i s probably more r e l e v a n t to the b i o l o g y of cancer i n humans than single-dose p r o t o c o l s . In a landmark study, Margison and Kleihues (1975) measured the l e v e l s of 0^-methylguanine i n the DNA of r a t t i s s u e s during weekly i n j e c t i o n s of MNU; a regime which s e l e c t i v e l y induces nervous system tumors. During 5 weekly i n j e c t i o n s of MNU, 0^-methylguanine accumulated i n the b r a i n to an extent g r e a t l y exceeding t h a t of the kidney, spleen, i n t e s t i n e and l i v e r . The l a t t e r organ had l e s s than 1% the 0^-methylguanine compared to the b r a i n . A l s o , between the f i r s t and f i f t h i n j e c t i o n s the r a t i o of 0^-methylguanine to 7-methylguanine (the major methyl adduct) i n c e r e b r a l DNA increased from 0.20 to 0.68. 15 0^-methylguanine has been connected to t r a n s f o r m a t i o n i n t i s s u e - c u l t u r e systems. S y r i a n hamster embryo c e l l s t r e a t e d w i t h methyl methanesulphonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and MNU were assayed f o r t r a n s f o r m a t i o n and i n d u c t i o n / r e p a i r of 0^-methylguanine (Doniger et a l . 1985). On a molar b a s i s , MNNG was approximately 100 and 500 f o l d more e f f e c t i v e i n t r a n s f o r m a t i o n than MNU and MMS r e s p e c t i v e l y . The formation of 0^-methylguanine was the same f o r the 3 carcinogens at concentrations t h a t induced e q u i v a l e n t t r a n s f o r m a t i o n frequencies. For MMS, however the i n d u c t i o n of 7-methylguanine was 3 0 - f o l d higher. 6.5 The Molecular Mechanism of Mutagenesis by 0^-methylguanine Research i n the past ten years has provided s e v e r a l l i n e s of evidence th a t s t r o n g l y suggest 0^-methylguanine i s r e s p o n s i b l e f o r the v a s t m a j o r i t y of gene mutations produced i n mammalian c e l l s by methylating agents (Pegg and Singer 1984). 6.5.1 I n V i t r o O x y g e n -alkylation of guanine r e s u l t s i n the deprotonation of N^ and the modif i e d base can no longer recognize c y t o s i n e (Parthasarathy and F r i d e y 1986) . Biochemical s t u d i e s have shown that thymidine or u r i d i n e gets i n c o r p o r a t e d p r e f e r e n t i a l l y opposite t h i s adduct i n co-polymers f o r E. coli polymerase I (Abbott and S a f f h i l l 1979) or RNA polymerase (Gerchman and Ludlum 1973). A l s o , 0^-methyldeoxyguanosine 5'-triphosphate i s p r e f e r e n t i a l l y i n c o r p o r a t e d a g a i n s t thymine on a template of calf-thymus DNA ( H a l l and S a f f h i l l 1983) or pBR322 DNA (Toorchen and Topal 1983). Therefore, a l k y l a t e d guanine acts as an adenine as f a r as base p a i r i n g i s concerned and recognizes thymine. 16 6.5.2 In Vivo 6.5.2.1 S i t e - d i r e c t e d Mutagenesis Recently, the advent of molecular techniques has enabled the genetic consequences of O^-methylguanine s p e c i f i c a l l y i n c o r p o r a t e d i n a known p o s i t i o n i n the genome to be assessed. In one case, DNA a n a l y s i s of 60 mutant v i r a l genomes r e v e a l e d t h a t O^-methylguanine induced e x c l u s i v e l y G -+ A t r a n s i t i o n mutations (Loechler et a l . 1984). E. coli c a r r y i n g the xanthine guanine p h o s p h o r i b o s y l t r a n s f e r a s e (gpt) gene on a pSV2gpt plasmid were exposed i n v i v o to MNU (Richardson et a l . 1987) . Using t h i s forward mutation system and di-deoxy c h a i n t e r m i n a t i n g DNA sequence methods, the type and frequency of s p e c i f i c DNA base changes (mutations) were compared to the l e v e l of O^-methylguanine i n the b a c t e r i a l genome. I t was found that 100% of MNU induced mutations were G/C -+ A/T t r a n s i t i o n s and 82% of the changes occurred at the middle guanine of the sequence 5'-GG(A/T)-3'. In v i v o , O^-methylguanine d i r e c t e d to a s p e c i f i c l o c a t i o n i n <f>X17h phage r e p l i c a t i n g form DNA has a mutagenic e f f i c i e n c y of 75% due e x c l u s i v e l y to thymidine m i s i n c o r p o r a t i o n s (Bhanot and Ray 1986). 6.5.2.2 Studies Comparing the L e v e l of O^-methylguanine Adduction and the  Frequency of S p e c i f i c Locus Mutation i n Mammalian C e l l s Newbold and co-workers reported the frequency of i n d u c t i o n of 8-azaguanine r e s i s t a n c e and oubain r e s i s t a n c e i n Chinese hamster V79 c e l l s t r e a t e d w i t h MMS, dimethy1sulphate, and MNU (Newbold et a l . 1980). They found t h a t the mutagenicity r e f l e c t s the c a r c i n o g e n i c i t y of these compounds and that d i f f e r e n c e s i n mutagenicity are p a r a l l e l e d by d i f f e r e n c e s i n l e v e l s of 0^-methylguanine. I n these r e p a i r d e f e c t i v e c e l l s , they observed a h i g h 17 c o r r e l a t i o n between the co n c e n t r a t i o n of 0^-methylguanine and the frequency of mutation. Suspensions of r e p a i r d e f e c t i v e Chinese hamster ovary c e l l s were exposed to MMS or MNU and assayed f o r mutation and a v a r i e t y of DNA adducts i n c l u d i n g 6 f> 0 -methylguanine (Beranek et a l . 1983). Only 0 -methylguanine c o r r e l a t e d s t r o n g l y (r=0.879, p r o b a b i l i t y l e s s than 0.001) w i t h the frequency of mutation at the hypoxanthine-guanosine phosphoribosyl t r a n s f e r a s e (HGPRT) lo c u s . 6.6 I n i t i a t i o n of Carcinogenesis through A c t i v a t i o n of C e l l u l a r Proto- oncogenes by 0^-methylguanine Induced Mutation There i s growing consensus that c e l l u l a r proto-oncogenes are the genetic t a r g e t s f o r i n i t i a t i o n of carcinogenesis by chemicals (Barbacid 1986, Bishop 1987). For example, i t i s now b e l i e v e d t h a t a c t i v a t i o n of H-ras proto-oncogenes by p o i n t mutations i s a r e s u l t of DNA damage produced by a carcinogen r a t h e r than as a consequence of the tr a n s f o r m a t i o n process (Bohr et a l . 1987). The r o l e of MNU induced 0^-methylguanine mutation of the H-ras-1 oncogene i n a r a t mammary carcinogenesis model has been reported r e c e n t l y ( Z a r b l et a l . 1985). A h i g h percentage of r a t s administered a s i n g l e dose of MNU develop mammary carcinomas. I t was found that 83% of the ch e m i c a l l y induced tumors contained an a c t i v a t e d H-ras-1 locus as determined by two independent methods (NIH 3T3 tr a n s f o r m a t i o n and r e s t r i c t i o n fragment l e n g t h polymorphisms). A l l of the a c t i v a t e d oncogenes had a G/C -• A/T t r a n s i t i o n at the middle guanine of codon 12 (5'-GGA-3'). This o b s e r v a t i o n supports the author's con t e n t i o n that mutations are caused by MNU d i r e c t e d methylation of the 0^ p o s i t i o n of the second guanine i n codon 12. This i s e x a c t l y what would be p r e d i c t e d i f 0^-methylguanine were r e s p o n s i b l e f o r mutation and n e o p l a s t i c transformation. 18 I n the r a t mammary tumor model, cancer i s achieved by a s i n g l e dose of MNU. Due to the h i g h l y l a b i l e nature of MNU, adduct formation must occur w i t h i n minutes of i t s a d m i n i s t r a t i o n . The r e s u l t s imply t h a t malignant a c t i v a t i o n of the Ha-ras-1 locus by MNU induced G -* A mutation i s concomittant w i t h i n i t i a t i o n of c a r c i n o g e n e s i s . Human ras oncogenes have been found to be produced by a m o d i f i c a t i o n of a G -* A t r a n s i t i o n or by a G -* T t r a n s v e r s i o n o c c u r r i n g at the same p o s i t i o n (codon 12) as the transforming H-ras-1 gene i n mammary carcinomas induced by MNU. 7. U l t r a - s e n s i t i v e Methodology f o r the D e t e c t i o n of ofi-methylguanine DNA  Adducts The i n f o r m a t i o n presented above supports the c u r r e n t hypothesis t h a t 0^-methylguanine i s the c r i t i c a l molecular l e s i o n whose formation i s necessary but probably not s u f f i c i e n t f o r the i n i t i a t i o n of ca r c i n o g e n e s i s by some N-nitrosamines (Pegg 1984). Methods f o r the d e t e c t i o n of 0^-methylguanine i n DNA i n c l u d e : a) HPLC fo l l o w e d by s c i n t i l l a t i o n counting a f t e r a d m i n i s t r a t i o n of l a b e l e d carcinogens, b) HPLC coupled w i t h fluorescence d e t e c t i o n and c) radioimmunoassays. Only the l a t t e r two are a p p l i c a b l e to human s t u d i e s . A t y p i c a l s e n s i t i v i t y f o r the HPLC/fluorescence assay i s 0.6 /imole/mole u s i n g 0.26 mg DNA (Herron and Shank 1979, Swenberg and B e d e l l 1982). The s e n s i t i v i t y of t h i s assay i s l i m i t e d by the fluorescence at s i m i l a r wavelengths of other compounds which may co-elute w i t h 0^-methylguanine on HPLC. Of the a v a i l a b l e methods, only immunoassays based on monoclonal a n t i b o d i e s appear s u f f i c i e n t l y s e n s i t i v e to detect 0 -methylguanine i n exposed humans (Wild et a l . 1983, W i l d et a l . 1986, Castonguay et a l . 1985). High s e n s i t i v i t i e s are achieved through HPLC recovery of 0^-methyldeoxyguanosine 19 from s e v e r a l mg d i g e s t e d DNA before antibody b i n d i n g . With these techniques the a b i l i t y to detect low l e v e l s of adduct i s l i m i t e d only by the amount of DNA a v a i l a b l e f o r h y d r o l y s i s . The h i g h e s t s e n s i t i v i t y f o r a radioimmunoassay r e p o r t e d i n the l i t e r a t u r e i s 0.008 /xmole/mole f o r HPLC a n a l y s i s of 1 mg d i g e s t e d DNA (Umbenhauer et a l . 1985). Recently a very h i g h a f f i n i t y murine monoclonal antibody g i v i n g 50% i n h i b i t i o n w i t h 1 fmole 0 -methyldeoxyguanosine has been reporte d (Parsa et a l . 1987). I f t h i s amount of adduct arose from 1 mg DNA the s e n s i t i v i t y would be 0.0003 /xmole/mole. 8. Current Problem Exposure of humans to N-nitrosamines i s a s s o c i a t e d w i t h up to 30% of a l l cancer deaths i n North America and i s a s i g n i f i c a n t c o n t r i b u t o r to disease i n many c o u n t r i e s (Bartch and Montesano 1984). The p r i n c i p a l mode of exposure i s through v o l u n t a r y l i f e s t y l e p r a c t i c e s , e s p e c i a l l y tobacco smoking and chewing (Hoffman and Hecht 1985, World Health O r g a n i z a t i o n 1985). Other sources are the food, work and l i v i n g environments (Singer et a l . 1986, IARC 1978, Moloney et a l . 1985). The b u l k of car c i n o g e n i c a c t i v i t y i n tobacco smoke and chewing tobacco e x t r a c t s i s due to a c l a s s of compounds known as tobacco s p e c i f i c N-nitrosamines (Hoffman and Hecht 1985). Many of these agents are capable of forming 0 -methylguanine i n the c e l l u l a r DNA of exposed i n d i v i d u a l s . The measurement of 0^-methylguanine i n e x f o l i a t e d c e l l s from s u s c e p t i b l e t i s s u e s may be s i g n i f i c a n t due to the mutagenic p o t e n t i a l of t h i s adduct and the strong c o r r e l a t i o n of DNA oxygen a l k y l a t i o n w i t h the car c i n o g e n i c potency of N-nitrosamines. To achieve h i g h s e n s i t i v i t i e s , immunoassays f o r 0^-methylguanine r e q u i r e m i l l i g r a m q u a n t i t i e s of DNA and are t h e r e f o r e not a p p l i c a b l e to the a n a l y s i s of s m a l l samples of e x f o l i a t e d c e l l s from i n d i v i d u a l s . C r o s s - r e a c t i v i t y of even monoclonal a n t i b o d i e s w i t h other adducts reduces the s e l e c t i v i t y of t h i s 20 method. There i s a requirement f o r an a l t e r n a t i v e method able to detect low l e v e l s of 0^-methylguanine i n microgram q u a n t i t i e s of DNA. 9. Ob]ectives The goals of t h i s p r o j e c t were: 32 1. To develop a P - p o s t l a b e l i n g assay f o r the d e t e c t i o n and q u a n t i t a t i o n of 0 -methylguanine adducts i n microgram q u a n t i t i e s of DNA. Method development proceeded c h r o n o l o g i c a l l y i n two stages: a) Synthesis and c h a r a c t e r i z a t i o n of 0^-methyldeoxyguanosine 3'-monophosphate (0 6mdG3'p). b) Development of chromatographic procedures f o r the i s o l a t i o n o f 06mdG3'p ft 32 from d i g e s t e d DNA and q u a n t i t a t i o n of 0 -methyldeoxyguanosine 3',5' P-bisphosphate (06mdG3'5'p) a f t e r p o s t l a b e l i n g . 2. To evaluate the performance of the method and a s c e r t a i n the f e a s i b i l i t y of i t s a p p l i c a t i o n to molecular epidemiology s t u d i e s i n humans exposed to N-nitrosamines. 3. To detect and q u a n t i t a t e 0^-methylguanine i n the DNA of mammalian c e l l s t r e a t e d w i t h a carcinogen (MNU) known to form t h i s adduct, u s i n g the new methodology. 21 MATERIALS 1. Chemicals and Enzymes Deoxyguanosine was purchased from Boehringer Mannheim, Dorv a l , Quebec. Diazomethane i n ether was prepared by d i s t i l l a t i o n of an a l k a l i n e s o l u t i o n of N-methyl-N-nitrosotoluenesulfonamide ( D i a z a l d , Sigma Chemical Company). The d i s t i l l a t i o n apparatus was from the A l d r i c h Chemical Company. Anhydrous KI^PO^ (ACS) was purchased from F i s h e r S c i e n t i f i c Company. R e d i s t i l l e d formamide was from Bethesda Research L a b o r a t o r i e s . L y o p h y l i z e d snake venom, 32 nuclease P I , and reagents f o r the p r e p a r a t i o n of P-ATP were from Boehringer Mannheim, Dorv a l , Quebec or from the Sigma Chemical Company, St. Lou i s , MO. P o l y n u c l e o t i d e k i n a s e , cloned, was purchased from U.S. Biochemicals, Cleveland, OH. Proteinase K (Protease Type X I ) , ribonuclease-A (Type 1-A from bovine pancreas), micrococcal nuclease, spleen phosphodiesterase, and potato apyrase were purchased from the Sigma Chemical Company, St. L o u i s , MO. 2-Ethoxyethanol was from BDH Chemicals, Toronto, Ontario. Undenatured ethanol (100%) f o r DNA p r e c i p i t a t i o n s was d i s t i l l e d twice i n g l a s s . Phenol, f o r DNA e x t r a c t i o n s , was g l a s s - d i s t i l l e d , e q u i l i b r a t e d w i t h T r i s - C l b u f f e r pH 8.0, and s t o r e d f r o z e n at -20°C before use. I s o b u t y r i c a c i d was from BDH Chemicals L t d . , Poole, England. MNU was from the Sigma Chemical Company, St. Lou i s , MO. and contained 25% by weight of a 3% a c e t i c a c i d s o l u t i o n as a p r e s e r v a t i v e . A l l other chemicals were reagent grade or b e t t e r . 2. Equipment U.V. s p e c t r a were obtained on a Lambda 3 UV/VIS spectrophotometer (Perkin-Elmer C o r p o r a t i o n ) . The 'multi-tube' v o r t e x e r (Model 2600) was from S c i e n t i f i c Manufacturing I n d u s t r i e s . Four ml polypropylene tubes were purchased from Elkay Products, Inc., of Shrewsbury, MA. Heat f o r the 22 ph o s p h o r y l a t i o n was provided by a 'Reacti-Therm' h e a t i n g module ( P i e r c e Chemical Company, Rockford, 111). Kodak 'X-Omat' XAR-5 X-ray f i l m (8 X 10 in) was exposed to chromatograms i n Kodak 'X-Omatic' c a s s e t t e s (8 X 10 i n ) equipped w i t h two Dupont Cronex ' l i g h t e n i n g - p l u s ' i n t e n s i f y i n g screens. Chromatograms were marked w i t h phosphorescent i n k u s i n g an 'Ultemit' autoradiography marker purchased from NEN Research Products. 3. Chromatography HPLC columns f o r the chromatography of deoxynucleoside 3'-monophosphates, deoxynucleoside 5'-monophosphates, and deoxynucleoside 3',5'-bisphosphates were custom packed by A l l t e c h , D e e r f i e l d , I I . They were 2.1 mm i n s i d e diameter X 100 mm and contained 5 micron ODS-3 reverse phase packing manufactured by Whatman, C l i f t o n , NJ. Column eluant was monitored at 260 nm us i n g a Beckman 165 TJV abso r p t i o n detector coupled to a Spectra-Physics SP4270 computing i n t e g r a t o r . A Perkin-Elmer 2/2 HPLC pumping system was used to produce high-pressure s o l v e n t . 1 M ammonium formate pH 3.5 ( f o r p r e p a r a t i o n of HPLC sol v e n t ) was made from g l a s s d i s t i l l e d formic a c i d and p u r i f i e d ammonium hydroxide. One mole formic a c i d i n 800 ml dcU^O was t i t r a t e d w i t h ammonium hydroxide to pH 3.3, then the s o l u t i o n cooled to room temperature. The t i t r a t i o n to pH 3.5 was then completed and the volume made up to one l i t e r . Reagent grade ammonium hydroxide and formic a c i d were used to prepare 2.25 M ammonium formate pH 3.5 f o r TLC. Ammonium formate s o l u t i o n s f o r b u f f e r i n g of DNA d i g e s t s and HPLC were made u s i n g g l a s s d i s t i l l e d formic a c i d and ammonium hydroxide. HPLC f r a c t i o n s were d r i e d down usin g a c e n t r i f u g a l vacuum evaporator (Model SVC 100H, Savant Corp., H i c k s v i l l e , N.Y.). PEI c e l l u l o s e t h i n l a y e r chromatography sheets (20 X 20 cm) were from Mackery Nagel, Germany and purchased through Brinkmann Instruments, Rexdale, 23 Ont. C e l l u l o s e coated TLC p l a t e s (20 X 20 cm, 0.1 mm l a y e r t hickness) were from E. Merck, Darmstadt, W. Germany and purchased through BDH, Toronto, Ontario, Canada. 4. T i s su e - c u l t u r e The CHO c e l l l i n e ( w i l d type) was obtained from Dr. Lome Whorton, U n i v e r s i t y of Toronto, Canada. These c e l l s are a subclone of the l i n e o r i g i n a l l y d e s c r i b e d by Kao and Puck (Gen. 55; 513-518, 1967). They were c u l t u r e d i n Eagle's Minimum E s s e n t i a l Medium (MEM) c o n t a i n i n g Hank's s a l t s supplemented w i t h 7.5% NaHCO-j, 10% f e t a l c a l f serum and a n t i b i o t i c s (kanamycin, fungizone, p e n i c i l l i n , and streptomycin). MNU was d i s s o l v e d i n 'wash' MEM which c o n s i s t e d of the above des c r i b e d 'growth' medium without f e t a l c a l f serum, kanamycin, and fungizone. The t i s s u e - c u l t u r e f l a s k s (175 2 9 cm f o r CHO experiments and 80 cm f o r C3H10T1/2 experiments) were purchased from Nunclon (Nunc, Intermed). The c e l l s were t r y p s i n i z e d u s i n g 0.1% t r y p s i n i n PBS. Mouse-embryo f i b r o b l a s t c e l l s (C3H10T1/2 Clone 8) were obtained from the American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , Maryland. These c e l l s are a subclone of a l i n e of C3H mouse embryo c e l l s i n i t i a t e d by C. R e z n i k o f f , D. Brankow, and C. Heidelberger i n 1972 (Cancer Res. 33: 3231-3238, 1973). They were c u l t u r e d and exposed to MNU i n Eagle's Basal Medium (BME) supplemented w i t h 7.5% NaHCOg, 10% f e t a l c a l f serum, L-glutamine, and a n t i b i o t i c s ( p e n i c i l l i n , streptomycin, kanamycin, and fungizone). For the colony forming assay the c e l l s were grown i n 60 mm dishes (with g r i d ) obtained from Nunclon. 24 METHOD DEVELOPMENT 1. Synthesis of O^-methvldeoxyguanosine 3'-monophosphate  I n t r o d u c t i o n DNA c o n t a i n i n g O^-methylguanine residues should give 0^mdG3'p when di g e s t e d w i t h m i c r o c o c c a l nuclease and spleen phosphodiesterase. The s t r u c t u r e of t h i s compound i s shown i n Figure 3. I d e n t i f i c a t i o n and 6 32 q u a n t i t a t i o n of 0 -methylguanine by P - p o s t l a b e l i n g r e q u i r e d the a v a i l a b i l i t y of s y n t h e t i c 0^mdG3'p as a chromatography marker. No p u b l i s h e d s y n t h e s i s was a v a i l a b l e f o r t h i s compound. I n i t i a l l y , two routes were i n v e s t i g a t e d to prepare 0^mdG3'p. D i r e c t m e t h y l a t i o n of deoxyguanosine 3'-monophosphate w i t h diazomethane was u n s u c c e s s f u l , presumably because phosphate methylation predominates. Diazomethane met h y l a t i o n of DNA f o l l o w e d by enzymatic d i g e s t i o n to deoxynucleoside 3'-monophosphates f a i l e d , l i k e l y f o r the same reason. Phosphorylation of O^-methyldeoxyguanosine, f o r which there are many reported syntheses (Farmer et a l . 1973, Bernadou et a l . 1983), u s i n g P O C I 3 i n t r i e t h y l p h o s p h a t e gave l a r g e l y 0^-methylguanine through probable a c i d i c decomposition. Amide c a t a l y s e d condensation of deoxynucleosides w i t h orthophosphate s a l t s a t e l e v a t e d temperatures gives isomeric mixtures of deoxynucleoside monophosphates ( P h i l i p p and S e l i g e r 1977, S c h o f f s t a l l 1976, S c h o f f s t a l l and Kokko 1978, S c h o f f s t a l l e t a l . 1982, S c h o f f s t a l l and Laing 1984). A p r e l i m i n a r y experiment showed that r e a c t i o n of excess K^PO^ w i t h deoxyguanosine f o r 9 hours i n formamide at 100°C gives a major product that co-chromatographs w i t h deoxyguanosine 3'-monophosphate. 25 Figure 3 Chemical s t r u c t u r e of 0 mdG3'p. 26 Therefore, i t was decided to attempt the s y n t h e s i s of 0^mdG3'p i n a two stage f a s h i o n ; f i r s t , p r e p a r a t i o n of O^ mdG by diazomethane methylation of deoxyguanosine (Farmer et a l . 1973) then phosphorylation of the compound using KH^PO^ i n formamide. Methods and R e s u l t s 1.1 P r e p a r a t i o n of 0^-methyldeoxyguanosine  M e t h y l a t i o n of deoxyguanosine To a suspension of 95 mg deoxyguanosine (.356 mmole) i n 15 ml methanol was added 34 ml f r e s h l y prepared e t h e r e a l diazomethane (7.12 mmole). The r e s u l t i n g y e l l o w suspension was l o o s e l y stoppered then s t i r r e d at room temperature. A f t e r two hours most of the deoxyguanosine had reacted, some diazomethane remained and an i n s o l u b l e white p r e c i p i t a t e appeared. The mixture was then reduced to dryness by r o t a r y evaporation at 30°C. The residue l e f t behind was d i s s o l v e d i n 1.5 ml 0.1 M ammonium formate pH 3.5 c o n t a i n i n g 20% methanol to give a y e l l o w i s h s o l u t i o n . A sm a l l amount of i n s o l u b l e white m a t e r i a l was removed by c e n t r i f u g a t i o n . I n a d d i t i o n to O^mdG, and N" 7-methyldeoxyguanosine are major products of the m e t h y l a t i o n of deoxyguanosine w i t h diazomethane (Farmer e t a l . 1973). Of these components, O^ mdG should e l u t e l a s t from a reverse phase HPLC column (Abbott e t a l . 1980). When a small sample of the crude r e a c t i o n mixture was f r a c t i o n a t e d on a semi-prep column, four major peaks were seen ( F i g 4). The l a s t peak (15 min) was t e n t a t i v e l y i d e n t i f i e d by U.V. spectophotometry as O^ mdG (see below). 27 Figure 4 HPLC e l u t i o n p r o f i l e of isomeric methyldeoxyguanosines r e s u l t i n g from the methylation of deoxyguanosine w i t h e t h e r e a l diazomethane 28 I s o l a t i o n and P u r i f i c a t i o n of Q--methyldeoxyguanosine O^ mdG was i s o l a t e d and p u r i f i e d on a Whatman semi-preparative HPLC column (0.9 cm X 25 cm) c o n t a i n i n g 10 micron ODS-3 reversed phase packing. Ammonium formate ( 0.1 M, pH 3.5) c o n t a i n i n g 20% methanol was pumped through the column a t 5 ml/min usi n g a P e r k i n Elmer pumping system. Eluant was monitored at 254 run u s i n g an ISCO (Instrumentation S p e c i a l i t i e s Company) Type 6 o p t i c a l u n i t coupled to an ISCO UA5 monitor. O^ mdG was recovered from 1500 /J.1 crude r e a c t i o n mixture by i n j e c t i n g 200 fil a l i q u o t s onto the column and c o l l e c t i n g the l a s t peak as i t e l u t e d . This f r a c t i o n (approximately 160 ml) was reduced to dryness a t room temperature u s i n g a c e n t r i f u g a l s p i n d r i e r . The w h i t i s h product remaining was d i s s o l v e d i n 2 ml dctt^O then a small p o r t i o n analysed by HPLC as a check f o r p u r i t y . S everal minor i m p u r i t i e s were present i n c l u d i n g one that e l u t e d j u s t a f t e r 06mdG. To remove t r a c e contaminants from O^mdG, a second HPLC p u r i f i c a t i o n was performed. I n 200 fil a l i q u o t s , the impure product was i n j e c t e d onto the column and the m a j o r i t y of the O^ mdG peak c o l l e c t e d . An im p u r i t y that co-e l u t e d w i t h the peak t a i l was excluded ( F i g 5). The f r a c t i o n (approximately 108 ml) was d r i e d down at room temperature u s i n g the c e n t r i f u g a l s p i n - d r i e r , then the p u r i f i e d O^ mdG d i s s o l v e d i n 2 ml dctt^O and analysed by HPLC ( F i g 6). I d e n t i f i c a t i o n of O^-methyldeoxyguanosine A U.V. spectrum of the product ( F i g 7) was i n c l o s e agreement to the l i t e r a t u r e values f o r O^ mdG (Farmer e t a l . 1973). Observed absorbance maxima (nm): 246, 278.5; minima: 224, 259. L i t e r a t u r e values f o r maxima: 247, 278; minima: 260. The y i e l d of 06mdG a f t e r HPLC recovery was 27.3 mg (27.3% of t h e o r e t i c a l ) . 29 1.8T a I . O H 0 6-mdG 0-0 t r ~8 impurity collect —i— 12 i— 16 20 Time (min) Figure 5 HPLC chromatogram of once p u r i f i e d 0 mdG. The product was p u r i f i e d a second time by c o l l e c t i n g the f r o n t p o r t i o n of the peak to exclude a slower running impurity. 30 Figure 6 HPLC a n a l y s i s of twice p u r i f i e d 0 mdG. 31 Figure 7 U.V. spectrum of 0°radG i n ddHjO. Observed absorbance maxima (nm) 246, 278.5. Minima: 224, 259. L i t e r a t u r e values (Farmer et a l . 1973) f o r maxima (nm): 247, 278. Minima: 260. 32 1.2 P r e p a r a t i o n of 0 -methvldeoxvguanosine 3'-monophosphate c Reaction of 0 -methvldeoxyguanosine w i t h Potassium Phosphate i n Formamide To 13.8 mg p u r i f i e d 0 mdG (.049 mmole) i n a 4 ml polypropylene tube, was added 134.8 mg anhydrous KI^PO^ (1 mmole) and 2.4 ml formamide (60.4 mmole). On thorough mixing, some Kr^PO^ and a l l of the O^ mdG d i s s o l v e d i n the formamide. The tube was then stoppered and the mixture heated to 100°C i n a hea t i n g block. To monitor the phosphorylation r e a c t i o n , the r e a c t i o n mixture was re s o l v e d on an a n a l y t i c a l reverse-phase HPLC column (Brownlee l a b s ) . A guard column (2.1 mm X 30 mm) was connected i n s e r i e s w i t h the main column (2.1 mm X 100 mm) and e l u t e d at 0.5 ml/min w i t h 0.1 M ammonium formate pH 3.5 co n t a i n i n g 10% methanol. On t h i s column the mono- and diphosphates of O^ mdG would be expected to e l u t e much e a r l i e r than the parent a l k y l a t e d nucleoside (Dunn, B. personal communication). A f t e r 1.5 hours h e a t i n g , 1 pil of the r e a c t i o n mixture was analysed by HPLC. Compared to a chromatogram of the mixture before h e a t i n g ( F i g 8A), a broad peak appeared t h a t ran f a s t e r than O^ mdG ( F i g 8B) and increased i n area w i t h time. The peak reached i t s maximum s i z e a f t e r nine hours. I s o l a t i o n and P u r i f i c a t i o n of P u t a t i v e Q--methvldeoxvguanosine 3'- and 5'- monophosphates When the phospho r y l a t i o n mixture was f r a c t i o n a t e d on a semi-preparative reverse-phase column the new peak was r e s o l v e d i n t o two separate peaks which were t e n t a t i v e l y designated as the monophosphates of O^ mdG ( F i g 9). The phosphorylated products were recovered together from the crude r e a c t i o n mixture by i n j e c t i n g 100 y,l a l i q u o t s onto the semi-preparative HPLC and c o l l e c t i n g the appropriate f r a c t i o n . Solvent was removed i n the s p i n - d r i e r 33 Figure 8 HPLC a n a l y s i s of 0°mdG/formamide/KH2P04 p h o s p h o r y l a t i o n mixture before (A) and a f t e r (B) 1.5 hours h e a t i n g a t 100°C. collect I 1 1 1 1 1 1 < 1 r 0 4 8 12 16 Time (min) Figure 9 P u t a t i v e 0 mdG monophosphates i n crude p h o s p h o r y l a t i o n mixture, separated by semi-preparative HPLC. Both peaks were c o l l e c t e d from 100 >il i n j e c t i o n s of the r e a c t i o n mixture. 35 then the residue d i s s o l v e d i n 1.5 ml dctt^O. Figure 10 shows an HPLC chromatogram of the p u r i f i e d products. For reverse-phase HPLC of n u c l e o t i d e s , the 5' isomers g e n e r a l l y run f a s t e r than the corresponding 3' isomers (Ramos and S c h o f f s t a l l 1983). Therefore, f o r purposes of d i s c u s s i o n , the f i r s t peak (Figure 10) was t e n t a t i v e l y designated as the 3'-monophosphate of O^mdG. The isomeric monophosphates were i n d i v i d u a l l y i s o l a t e d from the mixture by i n j e c t i o n of 100 nl a l i q u o t s onto the semi-preparative HPLC f o l l o w e d by separate c o l l e c t i o n of each peak. Both f r a c t i o n s were d r i e d down then each of the p u r i f i e d products d i s s o l v e d i n 1.5 ml dctt^O. The i s o l a t e d monophosphates were i s o m e r i c a l l y pure as determined by HPLC ( F i g 11). Based on U.V. abso r p t i o n , the y i e l d s of the p u t a t i v e 3'- and 5'- monophosphates a f t e r HPLC i s o l a t i o n were 3.04 pinole (6.2%) and 3.86 //mole (7.9%) r e s p e c t i v e l y . I d e n t i f i c a t i o n of 0^-methyldeoxyguanosine 3'-monophosphate I d e n t i t y of the p u t a t i v e 0^mdG3'p was confirmed by measurement of i t s U.V. spectrum and a n a l y s i s of r e a c t i o n products a f t e r exposure to enzymes w i t h known s p e c i f i c a c t i v i t i e s . The U.V. spectrum of p u t a t i v e 06mdG3'p ( F i g 12) and 06mdG5'p (not shown) are s i m i l a r to tha t of O^ mdG ( F i g 7). Deoxynucleoside 5'-monophosphates are hydrolysed to in o r g a n i c phosphate and deoxynucleoside by the 5'-nucleotidase of C. adamanteus venom (Sulkowski et a l . 1963, E t a i x and Orgel 1978). Deoxynucleoside 3'-monophosphates are r e s i s t a n t to t h i s a c t i v i t y . HPLC a n a l y s i s r e v e a l e d t h a t p u t a t i v e 0^mdG3'p was only s l i g h t l y decomposed to O^ mdG by treatment w i t h l y o p h i l i z e d venom ( F i g 13). P a r t i a l h y d r o l y s i s was probably caused by n o n - s p e c i f i c phosphatases present as i m p u r i t i e s i n the crude venom. 0^mdG5'p was completely converted to O^mdG by t h i s treatment (data not shown). 36 Figure 10 HPLC a n a l y s i s of isomeric 0 mdG3'and 5'-monophosphates i s o l a t e d from the r e a c t i o n mixture. 37 r i 1 i — » ' 1 1 1 r~ D 6 12 18 Time (min) Figure 11 HPLC e l u t i o n p r o f i l e s of p u r i f i e d 06mdG5'p (A) and 06mdG3'p (B). 38 0.05i Wavelength (nm) Figure 12 U.V. spectrum of 0°mdG3'p i n ddl^O. Observed absorbance maxima (nm): 248, 278. Minima: 228, 261. 39 Figure 13 C h a r a c t e r i z a t i o n of 0 mdG3'p usin g 5'-nucleotidase of C. adamanteus venom. Shown, are HPLC chromatograms of 0^mdG3'p before (A) and a f t e r (B) exposure to venom f o r 5 minutes. 40 Deoxynucleoside 3'-monophosphates are hydrolysed to i n o r g a n i c phosphate and deoxynucleoside by the 3'-nucleotidase a c t i v i t y of nuclease PI (Reddy et a l . 1984, Reddy and Randerath 1986). Deoxynucleoside 5'-monophosphates are r e s i s t a n t to t h i s enzyme. P u t a t i v e 0^mdG3'p was converted to O^ mdG by i n c u b a t i o n w i t h nuclease PI at 37°C f o r f i v e hours ( F i g 14). 0^mdG5'p was unchanged a f t e r the same treatment (data not shown). P o l y n u c l e o t i d e kinase c a t a l y s e s the t r a n s f e r of i n o r g a n i c phosphate from 32 the gamma p o s i t i o n of P-ATP to the 5' hydroxyl of deoxynucleoside 3'-monophosphates (Randerath e t a l . 1981). Deoxynucleosides and deoxynucleoside 5'-monophosphates are not substrates f o r t h i s enzyme and th e r e f o r e are not l a b e l e d . Incubation of p u t a t i v e 0^mdG3'p w i t h p o l y n u c l e o t i d e kinase and gamma 32 P-ATP gave r i s e to a l a b e l e d product t h a t migrated as a d i s t i n c t spot i n a 1-dimensional PEI-TLC system ( F i g 15). The l a b e l e d compound i s 06mdG3'5'p. 0^mdG5'p was not l a b e l e d under these c o n d i t i o n s . 2. 0 -Methylguanine Q u a n t i t a t i o n by P - P o s t l a b e l i n g A n a l y s i s I n t r o d u c t i o n Using s y n t h e t i c 0^mdG3'p as a chromatography marker, HPLC and TLC ( t h i n l a y e r chromatography) procedures were developed f o r the d e t e c t i o n and q u a n t i t a t i o n of 0 -methylguanine i n DNA by P - p o s t l a b e l i n g a n a l y s i s . HPLC was used to i s o l a t e 0^mdG3'p from DNA which had been di g e s t e d to deoxynucleoside 3'-monophosphates w i t h micrococcal nuclease and spleen 39 ft phosphodiesterase. I t was a l s o used to p u r i f y P-labeled 0 mdG3'5'p from r e s i d u a l l a b e l e d normal n u c l e o t i d e s and other r a d i o a c t i v e m a t e r i a l (method #2). To measure 0^-methylguanine i n DNA i t was necessary to r e s o l v e or separate 0^mdG3'5'p from other r a d i o a c t i v e compounds before s c i n t i l l a t i o n counting; t h i s was accomplished by PEI-TLC. To i n v e s t i g a t e enhancement of the 41 0.06^ LO C\J < 0 O.O61 B <M < 0 0 0-mdG3p 0 6-mdG "T 1 1 1 1 1 1 1 I 1 < l I I | | 1 | 2 4 6 8 Time (min) Figure 14 Conversion of 0°mdG3'p to 0°mdG by nuclease PI. HPLC chromatograms show 06mdG3'p before (A) and a f t e r (B) exposure to nuclease PI f o r 5 hours at 37°C. 42 Figure 15 1-Dimensional PEI-TLC autoradiogram of putative P-labeled 06mdG3'5'p derived from l a b e l i n g of 06mdG3'p with gamma J^P-ATP and polynucleotide kinase. D i r e c t i o n of solvent (2.25 M ammonium formate pH 3.5) migration i s bottom to top. 43 32 6 assays' performance, P - p o s t l a b e l i n g methodology f o r a n a l y s i s of 0 -methylguanine at both the 3',5'-bisphosphate and 5'-monophosphate l e v e l s was developed (methods #3 and #4). The four methods are summarized i n Figure 16 . Methods and R e s u l t s 2.1 Method #1: S i n g l e HPLC P u r i f i c a t i o n - D i p h o s p h a t e L e v e l  DNA E x t r a c t i o n DNA was i s o l a t e d from t i s s u e - c u l t u r e c e l l s u s i n g a m o d i f i c a t i o n of a p r e v i o u s l y d e s c r i b e d procedure (Maniatis et a l . 1982). 20-100 mg t i s s u e -c u l t u r e c e l l s were di g e s t e d f o r 3 hours at 37°C w i t h 500 /zg prot e i n a s e K i n 0.5 ml SET b u f f e r (100 mM NaCl, 20 mM EDTA, 50 mM T r i s - C l pH 8.0) c o n t a i n i n g 0.5% sodium dodecyl s u l f a t e (SDS). Samples were then s u c c e s s i v e l y e x t r a c t e d w i t h an equal volume of phenol, phenol/chloroform/isoamyl a l c o h o l (25:24:1), and chloroform/isoamyl a l c o h o l (24:1). The aqueous l a y e r was mixed w i t h the organic l a y e r u s i n g a 'multi-tube' v o r t e x e r , and i n t e r f a c e m a t e r i a l discarded. DNA was p r e c i p i t a t e d w i t h 2 volumes 2-ethoxyethanol and washed w i t h 1 ml c o l d 70% ethanol. To d i g e s t RNA, the DNA was r e d i s s o l v e d i n 0.5 ml SET c o n t a i n i n g 50 pg heat t r e a t e d p a n c r e a t i c r i b o n u c l e a s e , and incubated f o r 1 hour at 37°C. To remove r i b o n u c l e a s e , 50 fig proteinase K i n 10 fil SET b u f f e r was added to the tubes which were incubated at 37°C a f u r t h e r hour. The phenol, phenol/chloroform/isoamyl a l c o h o l , and chloroform/isoamyl a l c o h o l e x t r a c t i o n s were repeated then the DNA recovered by 2-ethoxyethanol p r e c i p i t a t i o n . To remove r e s i d u a l RNA, the DNA was d i s s o l v e d i n 0.25 ml SET b u f f e r and p r e c i p i t a t e d again w i t h 2 volumes 2-ethoxyethanol. This was repeated. F i n a l l y , the p u r i f i e d DNA was d i s s o l v e d i n 1 ml ddl^O and q u a n t i t a t e d by i t s 44 DNA micrococcal nuclease spleen phosphodiesterase dC3'p + dG3'p + dA3'p + dT3'p + 0°mdG3'p HPLC i s o l a t i o n 0°mdG3'p 32 32 P - l a b e l i n g P-ATP + T4 p o l y n u c l e o t i d e kinase 3 2 P - l a b e l e d 06mdG3'5'p remove ATP apyrase 3'-dephosphorylation nuclease PI Method #1 1. PEI-TLC 2. autorad. s c i n t i l l a t i o n HPLC 0°mdG3'5'p counting 1. c e l l - T L C 2. autorad. s c i n t i l l a t i o n counting 1. PEI-TLC 2. autorad. s c i n t i l l a t i o n counting Method #4 HPLC 0°mdG5'p 1. c e l l TLC 2. auto-rad . s c i n t i l l a t i o n counting On C F i g 16 Summary of the P - p o s t l a b e l i n g methods f o r 0 -methylguanine 45 U.V. a b s o r p t i o n a t 260 nm, usin g the r e l a t i o n s h i p of 1 absorbance u n i t = 50 Mg/ml (Sueoka and Cheng 1967) . RNA a s s o c i a t e d background r a d i o a c t i v i t y i n t e r f e r e s w i t h l a b e l e d 0^mdG3'5'p making q u a n t i t a t i o n p r o b l e m a t i c a l (see page 80 and Figure 26). P r i o r to p o s t l a b e l i n g a n a l y s i s the RNA content of the DNA was determined using a newly developed HPLC method (Dunn and San 1988). Preparations were f u r t h e r p u r i f i e d by ethoxyethanol p r e c i p i t a t i o n i f the contamination was s i g n i f i c a n t ( g r e a t e r than 5%). Enzymatic H y d r o l y s i s of DNA A l i q u o t s of DNA s o l u t i o n s each c o n t a i n i n g 1.5 jug DNA (about 4.5 nmol DNA n u c l e o t i d e s ) were reduced to dryness i n 1.5 ml polypropylene tubes us i n g a c e n t r i f u g a l vacuum evaporator. DNA was d i s s o l v e d i n 15 10 mM CaC^, 20 mM succ i n a t e b u f f e r pH 6.0 c o n t a i n i n g 3 ng (0.36 u n i t s ) m i c r o c o c c a l nuclease and 3 ng (0.006 u n i t s ) spleen phosphodiesterase then d i g e s t e d f o r 3 hours at 37°C (Randerath e t a l . 1981). This treatment hydrolyses the DNA to deoxynucleoside 3'-monophosphates. I s o l a t i o n of O^-methyldeoxyguanosine 3'-monophosphate by reverse-phase HPLC Reverse-phase HPLC was used to i s o l a t e femtomole ( 1 0 " ^ mole) q u a n t i t i e s of 0^mdG3'p from e n z y m a t i c a l l y digested DNA. The column was e l u t e d w i t h 95% 1 M ammonium formate pH 3.5 + 5% methanol at a flow r a t e of 0.4 ml/min. P r i o r to HPLC a n a l y s i s of DNA samples, 3 a l i q u o t s of 10 /*1 0.0905 mM deoxyadenosine 3'-monophosphate standard (0.905 nmole) were i n j e c t e d onto the column. This allowed determination of the t o t a l amount of DNA n u c l e o t i d e s a s s o c i a t e d w i t h each i n j e c t i o n . 46 In a t y p i c a l experiment, twelve DNA samples were analysed. DNA dig e s t s (15 / i l ) were mixed w i t h 15 /jI 2 M ammonium formate pH 3.5 and b r i e f l y c e n t r i f u g e d . For each sample, an i n j e c t i o n 'chase' of 10 (j.1 95% 1 M ammonium formate + 5% methanol was f i r s t drawn i n t o a HPLC syringe (50 //l capa c i t y ) f o l l o w e d by 20 of the d i l u t e DNA d i g e s t (about 3 nmoles DNA n u c l e o t i d e s ) to give a t o t a l i n j e c t i o n volume of 30 /il. A f t e r the sample was i n j e c t e d onto the column, the appropriate eluant f r a c t i o n c o n t a i n i n g 0^mdG3'p was c o l l e c t e d i n a 1.5 ml polypropylene tube (Figure 17). 6 6 Experiments w i t h s y n t h e t i c marker 0 mdG3'p showed th a t 0 mdG3'p t y p i c a l l y e l u t e d from the column i n a volume of 300 /il, 4.75-5.5 minutes a f t e r i n j e c t i o n . To guard against peak d r i f t caused by v a r i a b l e chromatographic c o n d i t i o n s , the a c t u a l c o l l e c t i o n f r a c t i o n was increased to 500 / / l (4.5-5.75 minutes). Solvent and b u f f e r were removed by vacuum evaporation i n a c e n t r i f u g a l s p i n - d r i e r a t room temperature f o r 12 hours. A f t e r sample DNA i n j e c t i o n s , a p o s i t i v e c o n t r o l c o n s i s t i n g of approximately 1 X 1 0 " ^ moles s y n t h e t i c 0^mdG3'p was chromatographed and analysed along s i d e the others. T h i s , and a sample composed of the same amount of 0^mdG3'p d i r e c t l y l a b e l e d without HPLC allowed c a l c u l a t i o n of the adduct recovery and subsequently, the adduct l e v e l s . 3 2 P - P o s t l a b e l i n g D r i e d residues from the evaporated HPLC f r a c t i o n s were d i s s o l v e d i n 5 nl 32 ddi^O. They were then P-labeled by adding 3 u n i t s of p o l y n u c l e o t i d e kinase, 1 Ml of kinase b u f f e r (0.2 M B i c i n e - C l pH 9.5, 0.1 M MgCl 2, 0.1 M 32 d i t h i o t h r e i t o l and 10 mM spermidine), 20 uCi gamma P-ATP and water to give a t o t a l volume of 10 /tl. A l l samples were incubated f o r 3 hours at 37°C (Dunn et a l . 1987). Excess P-ATP was converted to in o r g a n i c phosphate and ADP by treatment of the l a b e l e d d i g e s t w i t h 0.05 u n i t s apyrase ( i n 5 /zl 10 mM b i c i n e -47 0.2h AT o (0 CM < 0.1-6 0 -mdG3'p collection fraction i—i—i—i—i—i—» 2 4 6 Time (min) Figure 17 I s o l a t i o n of 0 mdG3'p from DNA dig e s t e d w i t h micrococcal nuclease and spleen phosphodiesterase u s i n g reverse-phase HPLC. The HPLC chromatogram i s from the a n a l y s i s of 1 jig methylated DNA (about 3 nmole DNA n u c l e o t i d e s ) c o n t a i n i n g 1 umole/mole 0 -methylguanine. C,G,T, and A are the 3'-monophosphates of deo x y c y t i d i n e , deoxyguanosine, thymidine, and deoxyadenosine r e s p e c t i v e l y . 48 CI pH 9.5 b u f f e r ) f o r 1/2 hour at room temperature (Gupta et a l . 1982). Samples were d i l u t e d to 100 p i w i t h 85 p i ddr^O and e i t h e r immediately chromatographed on P E I - c e l l u l o s e or s t o r e d overnight at -20°C. 32 P r e p a r a t i o n of Gamma --'P-ATP and Determination of i t s S p e c i f i c A c t i v i t y 32 P-ATP was syn t h e s i z e d on a weekly b a s i s , u s u a l l y two days before i t was to be used f o r l a b e l i n g . Using a p r e v i o u s l y described procedure, a c o c k t a i l of g l y c o l y t i c pathway enzymes c a t a l y s e d the r e a c t i o n between ADP and inorganic 32 to give the d e s i r e d product (Gupta et a l . 1982). The p r e p a r a t i o n was 32 managed so t h a t the c o n c e n t r a t i o n of P-ATP was 100 u C i / p l on the day of l a b e l i n g . Completeness of the r e a c t i o n was checked p r i o r to l a b e l i n g by one-dimensional TLC of the mixture on P E I - c e l l u l o s e . For p r e p a r a t i o n of the kinase r e a c t i o n mixture the P-ATP was d i l u t e d w i t h ddr^O. 32 For each batch of P-ATP made the s p e c i f i c a c t i v i t y was determined by l a b e l i n g f o u r 5 p i samples of 10"^ M deoxyadenosine 3'-phosphate (5 X 10-13 moles) (Reddy and Randerath 1986). The samples were chromatographed on PEI-c e l l u l o s e u s i n g 0.3 M ammonium s u l f a t e b u f f e r e d w i t h 10 mM Na phosphate pH 32 7.5. The spots c o n t a i n i n g P-labeled deoxyadenosine 3',5'-bisphosphate were cut out and t h e i r r a d i o a c t i v i t y measured by s c i n t i l l a t i o n counting (Dunn and 3 9 San 1988). Assuming 100% l a b e l i n g , the s p e c i f i c a c t i v i t y of P-ATP was c a l c u l a t e d and i t t y p i c a l l y had a value of 3000 Ci/mmole. This i s l e s s than the t h e o r e t i c a l f i g u r e of 9000 Ci/mmole and may be due to the presence of non-r a d i o a c t i v e i n o r g a n i c phosphate i n the mixture, d e r i v e d from one or more of the reagents used. 49 32 Q u a n t i t a t i o n of P-ATP Consumed during the Kinase Reaction To a c c u r a t e l y measure O^-methylguanine i n DNA by p o s t l a b e l i n g , a l l of the 0^mdG3'p present i n the HPLC f r a c t i o n must be l a b e l e d ; t h i s r e q u i r e s a molar 32 32 excess of P-ATP. In some assays, the amount of excess P-ATP l e f t a f t e r l a b e l i n g was monitored by a n a l y s i n g a small p o r t i o n of the r e a c t i o n mixture before apyrase treatment (Dunn and San 1988). This was accomplished by d i p p i n g the t i p of a wooden t o o t h p i c k i n t o the r e a c t i o n mixture then r i n s i n g the t o o t h p i c k i n 20 / i l water. A s m a l l a l i q u o t of the d i l u t e mixture (5 p i ) was then chromatographed on P E I - c e l l u l o s e i n one dimension u s i n g 0.3 M 32 ammonium sulphate. Unused P-ATP was estimated by comparing the r a d i o a c t i v i t y i n the P-ATP spot to the r a d i o a c t i v i t y i n the normal n u c l e o t i d e spots. Two-Dimensional P E I - C e l l u l o s e Thin Layer Chromatography 0°mdG3'5'p was r e s o l v e d from r e s i d u a l r a d i o a c t i v e normal n u c l e o t i d e s and other m a t e r i a l by two-dimensional t h i n l a y e r chromatography (TLC) on PEI-c e l l u l o s e . Commercially manufactured sheets were washed and marked before use. To remove i m p u r i t i e s from the sheets they were i n d i v i d u a l l y r i n s e d f o r 2 minutes i n methanol, then twice i n d e i o n i z e d water and f i n a l l y i n 0.225 M ammonium formate pH 3.5. Then, u s i n g a h a i r - d r y e r , the sheets were blown dry f o r 10 minutes. To f a c i l i t a t e o r i e n t a t i o n of the chromatograms w i t h the autoradiogram, the sheets were marked w i t h phosphorescent ink. The chromatographic o r i g i n (2 cm i n from the lower l e f t - h a n d corner) and d i r e c t i o n s of so l v e n t development were i n d i c a t e d on the sheets u s i n g a s o f t (#6B) p e n c i l . 50 The two-dimensional s o l v e n t system f o r chromatography of normal n u c l e o t i d e s used by Gupta and co-workers was modif i e d f o r optimal r e s o l u t i o n of 06mdG3'5'p from other spots (Gupta et a l . 1982). 10 / i l of the d i l u t e d r a d i o a c t i v e sample (10% of the l a b e l e d HPLC f r a c t i o n ) was s l o w l y spotted at the marked o r i g i n . A f t e r blow d r y i n g the spot, the chromatograms were developed to the top edge i n 2.25 M ammonium formate pH 3.5 (Dl) ; t h i s took about 1.5 hours. Chromatography was performed i n c l o s e d tanks w i t h a solvent depth of about 1 cm. A f t e r D l , ammonium formate was removed from the chromatograms u s i n g a cur r e n t of warm a i r from a h a i r dryer. Chromatograms were then soaked v e r t i c a l l y f o r 5 minutes i n d e i o n i z e d water then f o r 5 minutes i n 1 mM Na^PO^ pH 7.5. O r i e n t a t i o n during soaking was the same as f o r D2. A f t e r blow d r y i n g f o r 10 minutes the chromatograms were developed to the top edge i n 0.3 M ammonium sulphate + 0.01 M Na^PO^ pH 7.5. This run (D2 at r i g h t angles to Dl) a l s o r e q u i r e d about 1.5 hours. F i n a l l y , the sheets were blown dry f o r the next stage, autoradiography. Autoradiography and Measurement of R a d i o a c t i v i t y i n the 0^- methy1deoxyguanosine 3'.5'-bisphosphate spot Chromatograms were autoradiographed w i t h X-ray f i l m i n c a s s e t t e s equipped w i t h 2 i n t e n s i f y i n g screens f o r 2 hours a t room temperature (Dunn and S t i c h 1986). F i l m was then developed f o r 5 minutes i n Kodak D-19, immersed i n a stop bath f o r 1 minute then f i x e d u n t i l c l e a r (5-10 minutes). A f t e r r i n s i n g f i l m s i n running tap water f o r 10 minutes, they were d r i e d u s i n g an automatic r o l l e r d r i e r . Representative autoradiograms are shown i n Figure 18. Chromatographic c o n d i t i o n s i n the HPLC step were such t h a t 0^mdG3'p e l u t e d at about three times the r e t e n t i o n time of dA3'p, which was the l a s t normal n u c l e o t i d e . This r e s u l t e d i n traces of a l l f our normal n u c l e o t i d e s being gure 18 Representative autoradiograms f o r p o s t l a b e l i n g a n a l y s i s of 0 -methylguanine i n 1 }xg DNA usin g method #1 ( s i n g l e HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) . A. . DNA from untreated CHO c e l l s . B. DNA c o n t a i n i n g 55 ,umole/mole 0^-methylguanine from CHO c e l l s t r e a t e d w i t h MNU. C. P o s i t i v e c o n t r o l c o n s i s t i n g of 4 X 1 0 " ^ moles s y n t h e t i c 0^mdG3'p i n j e c t e d onto the HPLC. D. Blank sample c o n s i s t i n g of 500 ;ul HPLC so l v e n t c o l l e c t e d from column before DNA i n j e c t i o n s . G,A,C,T and 0 6 are the 3 2 P - l a b e l e d 3'5' bisphosphates of deoxyguanosine, deoxyadenosine, de o x y c y t i d i n e , thymidine and 06mdG r e s p e c t i v e l y . 52 c present i n the 0 mdG3'p f r a c t i o n , however these are w e l l r e s o l v e d from 06mdG3'5'p by 2-dimensional PEI-TLC ( F i g 18). With the a i d of a l i g h t box, the o u t l i n e o f the 06mdG3'5'p spot on the f i l m was t r a c e d onto the face of the TLC chromatograms u s i n g a s o f t p e n c i l . The c i r c l e d s e c t i o n of chromatogram was then punched out us i n g a 13 mm diameter ' a r t ' punch (Dunn and San 1988). The l e v e l of r a d i o a c t i v i t y i n the 0^mdG3'5'p spot was determined by l i q u i d s c i n t i l l a t i o n counting. The r a d i o a c t i v e chip was combined w i t h 5 ml toluene based s c i n t i l l a t i o n f l u i d i n a p l a s t i c screw top s c i n t i l l a t i o n v i a l . Samples were then counted f o r 10 minutes at a counting e f f i c i e n c y of e s s e n t i a l l y 100%. Q u a n t i t a t i o n of 0^-methylguanine The amount (moles) of deoxyadenosine 3'-monophosphate i n a di g e s t e d DNA sample t h a t was i n j e c t e d onto the HPLC was determined by comparison of i t s peak area w i t h t h a t of a known amount of standard analysed during the same chromatographic run. This number was d i v i d e d by 0.29 (the prevalence of adenosine i n DNA from v e r t e b r a t e s ) to give the t o t a l moles of DNA n u c l e o t i d e s a s s o c i a t e d w i t h each kinase r e a c t i o n (Sober 1968). The moles of 0^mdG3'5'p was c a l c u l a t e d by d i v i d i n g the s p e c i f i c a c t i v i t y of 3 2P-ATP i n t o the 06mdG3'5'p counts. To c o r r e c t f o r HPLC a s s o c i a t e d l o s s e s , t h i s value was d i v i d e d by the recovery (determined from the p o s i t i v e c o n t r o l ) . F i n a l l y , t h i s number was m u l t i p l i e d by ten to give the t o t a l moles of 0^mdG3'5'p s i n c e only 10% of the kinase r e a c t i o n was analysed by TLC. For a l l 32 analyses, the s p e c i f i c a c t i v i t y of P-ATP was determined on the same day as the 0^mdG3'5'p spots were counted. D i v i s i o n of moles of 0^mdG3'5'p by t o t a l moles DNA n u c l e o t i d e s f o r a sample gave the l e v e l of 0^-methylguanine i n the DNA. This was conveniently expressed as micromoles adduct per mole normal n u c l e o t i d e s (//mole/mole). 53 2.2 Method #2: Dual HPLC P u r i f i c a t i o n - D i p h o s p h a t e L e v e l c For f u r t h e r p u r i f i c a t i o n of 0 mdG3'5'p by reverse-phase HPLC, 15 p i HPLC c a r r i e r was added to the 15 p i apyrase t r e a t e d kinase r e a c t i o n (see page 46) and the mixture c e n t r i f u g e d b r i e f l y . HPLC c a r r i e r was 2 M ammonium formate pH 3.5 c o n t a i n i n g 2 mM of the 3',5'-bisphosphates of deoxy c y t i d i n e , thymidine and deoxyadenosine, and 10 mM potassium dihydrogen phosphate. Preceded by 10 p i of an HPLC s o l v e n t 'chase', a l l of the r a d i o a c t i v e s o l u t i o n was drawn up i n t o an HPLC syringe to give an i n j e c t i o n volume of 40 / t l per sample. This was then i n j e c t e d onto a reverse-phase HPLC column and e l u t e d w i t h 1 M ammonium formate pH 3.5 c o n t a i n i n g 1% methanol at a flow r a t e of 0.4 ml/min. The eluant f r a c t i o n c o n t a i n i n g 0^mdG3'5'p was c o l l e c t e d i n a 1.5 ml polypropylene tube and reduced to dryness f o r 12 hours at room temperature i n a c e n t r i f u g a l s p i n - d r i e r . 32 6 Experiments w i t h P-labeled s y n t h e t i c 0 mdG3'5'p under the same chromatographic c o n d i t i o n s showed that the bisphosphate t y p i c a l l y e l u t e d from the column i n a volume of 500 / t l , 3.75-5 minutes a f t e r i n j e c t i o n . To compensate f o r experimental v a r i a b i l i t y i n e l u t i o n p o s i t i o n s , the a c t u a l c o l l e c t i o n f r a c t i o n was increased to 700 p i (3.5-5.25 minutes). The d r i e d r a d i o a c t i v e residue was d i s s o l v e d i n 20 / i l 10 mM ammonium formate pH 3.5 and s l o w l y spotted a t the o r i g i n of a p r e - t r e a t e d P E I - c e l l u l o s e sheet marked f o r 2-dimensional TLC. The spot was blown dry then chromatography, autoradioagraphy (12 hours), and s c i n t i l l a t i o n counting performed as d e s c r i b e d p r e v i o u s l y . 54 2.3 Method #3: S i n g l e HPLC Purification-Monophosphate L e v e l 32 A f t e r P - p o s t l a b e l i n g of the HPLC f r a c t i o n (see page 46), 7.5 p i of nuclease PI i n b u f f e r ( c o n s i s t i n g of 1.5 p i 5 mg/ml nuclease PI i n water, 3.75 Hi of 0.25 M Na acetate pH 5.0, 2.25 /xl 0.3 mM Zn C l 2 ) was added to the 32 s o l u t i o n . This treatment converts P-labeled deoxynucleoside 3'5'-3? bisphosphates to the corresponding P-labeled deoxynucleoside 5'-monophosphates. Incubation was at 37°C f o r one hour. The sample was then d i l u t e d to 50 / i l by the a d d i t i o n of 21.5 / i l ddH 20 and 11 p i 'TLC c a r r i e r ' and st o r e d overnight a t - 20°C. 'TLC c a r r i e r ' was 2.5 mg each of ATP, and the 5'-monophosphates of deo x y c y t i d i n e , thymidine, deoxyguanosine and deoxyadenosine p l u s 150 / i l of 2.6 X 10" 3 M unlabeled 06mdG5'p d i s s o l v e d i n 500 p i ddH 20. Compared to deoxynucleoside bisphosphates, 2-dimensional TLC of 3 2 P -l a b e l e d 0^mdG5'p r e q u i r e s d i f f e r e n t s o l v e n t s and t h i n l a y e r chromatogram c h a r a c t e r i s t i c s . The 2-D TLC system employed by Wilson and co-workers to 32 q u a n t i t a t e 5-methylcytosine by P - p o s t l a b e l i n g a t the 5'-monophosphate l e v e l was t h e r e f o r e adapted f o r t h i s purpose (Wilson e t a l . 1986). C e l l u l o s e p l a t e s were marked f o r 2-dimensional TLC u s i n g a s o f t p e n c i l and f o r autoradiography u s i n g phosphorescent ink. The p l a t e was spotted w i t h 2 p i of the d i l u t e r a d i o a c t i v e sample a t the o r i g i n (2 cm from the lower l e f t -hand corner) and the spot allowed to a i r - d r y . The chromatograms were developed to the top edge (Dl) u s i n g i s o b u t y r i c a c i d : H 20: concentrated ammonium hydroxide (66:20:1). This run took about 10 hours. They were then d r i e d overnight i n a fumehood. Next day, Dl was repeated. On the t h i r d day, the chromatograms were turned 90° and run to the top edge (D2) i n saturated ammonium sulphate: isopropanol: 1 M sodium acetate (80:2:18). This run re q u i r e d about 9 hours. F i n a l l y the p l a t e s were a i r d r i e d overnight i n a fumehood. 55 The chromatograms were autoradiographed f o r 7 hours a t room temperature. The 0^mdG5'p spot was scraped o f f the p l a t e u s i n g a s p a t u l a and the r a d i o a c t i v i t y measured by s c i n t i l l a t i o n counting. T y p i c a l autoradiograms are shown i n Figure 19. 32 P-ATP migrated as a d i s t i n c t spot i n the two-dimensional TLC system used (see Figure 19). Measurement of r a d i o a c t i v i t y i n t h i s spot allowed 32 v e r i f i c a t i o n of excess P-ATP i n the kinase r e a c t i o n . A l t e r n a t i v e l y , using a to o t h p i c k , a small sample of the kinase r e a c t i o n was analysed a f t e r nuclease PI treatment, by 1-dimensional P E I - c e l l u l o s e TLC. 2.4 Method #4: Dual HPLC Purification-Monophosphate L e v e l To the 17.5 p i nuclease PI t r e a t e d kinase r e a c t i o n (see page 54) was added 17.5 p i 'HPLC c a r r i e r ' and the mixture b r i e f l y c e n t r i f u g e d . 'HPLC c a r r i e r ' was 1.5 M ammonium formate pH 3.5 c o n t a i n i n g 0.57 mM of the 5'-monophosphates of deoxy c y t i d i n e , thymidine, deoxyguanosine, deoxyadenosine, and O^-methyldeoxyguanosine plus 0.57 mM ATP. Along w i t h a 'chase' of 10 p i HPLC s o l v e n t the e n t i r e mixture was i n j e c t e d onto the column and e l u t e d w i t h 1 M ammonium formate pH 3.5 p l u s 1% methanol a t 0.4 ml/min. With the detector (254 nm) s e n s i t i v i t y s et at 0.5 AUFS, the peak corresponding to c o l d 0^mdG5'p was c o l l e c t e d i n a 1.5 ml polypropylene tube (Figure 20). Then, 1 p i of d i l u t e d TLC c a r r i e r (1:10 i n ddl^O) was added to the f r a c t i o n and b u f f e r / s o l v e n t removed by evaporation i n a c e n t r i f u g a l s p i n - d r i e r overnight at room temperature. A t y p i c a l c o l l e c t i o n f r a c t i o n f o r 0^mdG5'p was 4 minutes to 6 minutes 30 seconds a f t e r i n j e c t i o n (volume=l ml). This corresponded to a peak center e l u t i o n time of 4 minutes 30 seconds. The f r a c t i o n was r e l a t i v e l y l a r g e owing to the pronounced t a i l i n g of the peak. 56 T O C Figure 19 Representative autoradiograms f o r P - p o s t l a b e l i n g a n a l y s i s of 0 • methylguanine i n 1 >ig DNA usi n g method #3 ( s i n g l e HPLC purification-monophosphates). A. . DNA from untreated C3H10T1/2 mouse embryo f i b r o b l a s t c e l l s . B. 1 ug d i g e s t e d C3H10T1/2 DNA spiked w i t h 27.5 umole/mole s y n t h e t i c 06mdG3'p. C. P o s i t i v e c o n t r o l c o n s i s t i n g of 6.87 X 1 0 " ^ moles s y n t h e t i c 06mdG3'p i n j e c t e d onto the HPLC. D. Blank sample c o n s i s t i n g of 500 p.1 HPLC sol v e n t c o l l e c t e d from the column before DNA i n j e c t i o n s . 57 Figure 20 P u r i f i c a t i o n of J P-labeled 0 mdGS'p usin g reverse-phase HPLC (method #4). Cold 0 mdG5'p added to the nuclease PI t r e a t e d kinase mixture serves as a U.V. marker f o r the recovery of P-l a b e l e d 06mdG5'p. C. Peak due to unlabeled ATP and deoxynucleoside 5'-monophosphates i n 'HPLC c a r r i e r ' . 58 The r a d i o a c t i v e residue was d i s s o l v e d i n 10 / t l 10 mM ammonium formate pH 3.5. This s o l u t i o n was then spotted, i n 2 / i l a l i q u o t s w i t h d r y i n g i n between, onto the o r i g i n of a c e l l u l o s e TLC p l a t e marked f o r 2-dimensional TLC. Chromatography, autoradiography and s c i n t i l l a t i o n counting were performed as d e s c r i b e d p r e v i o u s l y . Sample autoradiograms are shown i n Figure 21. 32 Confirmation of excess P-ATP i n the kinase r e a c t i o n a f t e r nuclease PI treatment was e s t a b l i s h e d by assaying a small a l i q u o t of the r a d i o a c t i v e m a t e r i a l u s i n g a t o o t h p i c k as described p r e v i o u s l y . 39 ft 3. Performance of the ' P - P o s t l a b e l i n g Assays f o r 0 -methylguanine I n t r o d u c t i o n Three p r i n c i p a l determinants of an assay's performance are i t s r e p r o d u c i b i l i t y , recovery and s e n s i t i v i t y (Garner 1985, Lohman et a l . 1984). A measure of the r e p r o d u c i b i l i t y i s the standard d e v i a t i o n of adduct l e v e l s on r e p l i c a t e a n a l y s i s of a s i n g l e DNA sample. The recovery i s the f r a c t i o n of 0^mdG3'p r e l e a s e d on enzymatic d i g e s t i o n of DNA which i s f i n a l l y counted as l a b e l e d 0 mdG3'5'p on the TLC chromatograms, a f t e r passage through v a r i o u s HPLC/TLC p u r i f i c a t i o n s before and a f t e r p o s t l a b e l i n g . S e n s i t i v i t y r e f e r s to the lowest l e v e l of adduct which can be r e l i a b l y detected above background f o r a given amount of DNA analysed. 59 Figure 21 Sample autoradiograms f o r P - p o s t l a b e l i n g a n a l y s i s of 0 -methylguanine i n 1 pg DNA usin g method #4 (dual HPLC p u r i f i c a t i o n -monophosphate l e v e l ) . A. DNA from untreated C3H10T1/2 c e l l s . B. 1 >ig d i g e s t e d DNA spiked w i t h 17 >imole/mole s y n t h e t i c 06mdG3'p. C. Sample c o n s i s t i n g of d i r e c t l y l a b e l e d s y n t h e t i c 0 mdG3'p with no HPLC p u r i f i c a t i o n . P_. Assay blank created by the l a b e l i n g of 5 ^ i l ddH 20. 60 Methods and R e s u l t s 3.1 R e p r o d u c i b i l i t y of the Assay f o r Syn t h e t i c O mdG3'p HPLC of r e p l i c a t e i n j e c t i o n s of standard f o l l o w e d by p o s t l a b e l i n g of the f r a c t i o n , TLC a n a l y s i s and counting of the 0^mdG3'5'p spots can give an estimate of the assay r e p r o d u c i b i l i t y w i t h i n a s i n g l e sample of dig e s t e d DNA. This i s one source of v a r i a t i o n c o n t r i b u t i n g to the o v e r a l l assay r e p r o d u c i b i l i t y between s e p a r a t e l y d i g e s t e d a l i q u o t s from a s i n g l e DNA sample. F i r s t , a s o l u t i o n c o n s i s t i n g of 15 p i 2.83 X 10" 9 M 06mdG3'p and 15 fj.1 2 M ammonium formate pH 3.5 was prepared. Then, four 5 fil a l i q u o t s of t h i s s o l u t i o n were s e p a r a t e l y i n j e c t e d onto the HPLC and the appropriate f r a c t i o n c o l l e c t e d . 0^mdG3'p was measured i n the samples according to method #1. Values f o r the r a d i o a c t i v i t y i n the adduct spots are presented i n Table 1. From these data, the standard d e v i a t i o n f o r r e p l i c a t e a n a l y s i s of 0^mdG3'p from a s i n g l e sample i s + 6%. 3.2 R e p r o d u c i b i l i t y of DNA D i g e s t i o n An estimate of the r e p r o d u c i b i l i t y of enzymatic r e l e a s e of 0^mdG3'p from DNA may be obtained by determining the standard d e v i a t i o n of DNA nu c l e o t i d e y i e l d s from m u l t i p l e s e p a r a t e l y d i g e s t e d a l i q u o t s of a s i n g l e DNA sample using HPLC. The values f o r r e p l i c a t e d i g e s t i o n of 1 ng DNA are shown i n Table 2. For 10 experiments i n v o l v i n g separate DNA samples analysed a t d i f f e r e n t times, the standard d e v i a t i o n s of d i g e s t i o n range from + 0.9% to + 7.3%. 61 TABLE 1 REPRODUCIBILITY OF THE 3 2P-POSTLABELING ASSAY FOR SYNTHETIC O6-METHYLDEOXYGUANOSINE 3'-MONOPHOSPHATE Sample J ZP-0 mdG3'5'p R a d i o a c t i v i t y (cpm) 1 3411.0 2 3542.8 3 3932.0 4 :3686.0 Mean 3643.0 + 223 (SD) Note: R e p l i c a t e i n j e c t i o n s (1-4) of standard 0 mdG3'p on the reverse-phase HPLC column were analysed according to method #1. Values were c o r r e c t e d by s u b t r a c t i n g a column blank value of 6770.4 cpm. The column blank c o n s i s t e d of 500 nl s o l v e n t c o l l e c t e d from the column p r i o r to a c t u a l samples and analysed i n the same manner. 62 TABLE 2 REPRODUCIBILITY OF DNA DIGESTION i t e s ( n ) a Nucleotides (nmole)^ Standard D e v i a t i o n 10 2. .56 0. .07 10 2. .20 0. .16 10 2, .26 0. .05 4 2 .14 0, .08 4 2, .32 0. .02 6 2 .27 0. .05 4 2 .64 0, .03 7 2 .60 0 .18 4 2 .07 0 .08 4 2 .32 0 .03 Mean = 2.34 Mean = 0.08 Note: The t a b l e shows the r e s u l t s of 10 independent t r i a l s where 1 ng (determined by U.V.) a l i q u o t s of a s i n g l e DNA sample was s e p a r a t e l y d i g e s t e d u s i n g MN/SPD. Nucleotides were q u a n t i t a t e d by HPLC a n a l y s i s and measurement of the dA3'p peak. a Denotes the number (n) of 1 pg DNA a l i q u o t s d i g e s t e d f o r each t r i a l . k The mean y i e l d of DNA n u c l e o t i d e s from d i g e s t i o n of n a l i q u o t s of 1 pg DNA. 63 3.3 Recovery of O^-methyldeoxvguanosine 3'-monophosphate To determine the recovery of 0^mdG3'p from d i g e s t e d DNA, DNA from unexposed c e l l s was digested, then spiked w i t h a known amount of s y n t h e t i c marker, and analysed by p o s t l a b e l i n g . The HPLC recovery of 0 mdG3'p was determined by comparison of spot i n t e n s i t i e s between the p u r i f i e d DNA sample and one c o n s i s t i n g of the same amount of 0^mdG3'p d i r e c t l y l a b e l e d (no HPLC). In one experiment, 1.5 pg C3H10T1/2 DNA was di g e s t e d u s i n g micrococcal nuclease and spleen phosphodiesterase then spiked w i t h 8.42 X 1 0 " ^ moles 0^mdG3'p. 2/3 of t h i s mixture was i n j e c t e d onto the HPLC and the adduct subsequently q u a n t i t a t e d u s i n g method #1. At the same time, 5.61 X 10"-1-4 moles 0^mdG3'p (equal to the amount of standard i n the spi k e d DNA sample) was analysed by d i r e c t l a b e l i n g without HPLC p u r i f i c a t i o n . Comparison of spot i n t e n s i t i e s f o r d u p l i c a t e s of each type of sample gave a recovery of 81%. In a s i m i l a r experiment 1.5 pg dig e s t e d calf-thymus DNA was spi k e d w i t h a lower l e v e l of 06mdG3'p (1.58 X 1 0 " 1 4 moles). The recovery i n t h i s case was 88%. In two experiments without DNA i n v o l v i n g 1.5 and 1.7 X 1 0 m o l e s 0^mdG3'p, re c o v e r i e s were 86% and 82% r e s p e c t i v e l y . The recovery of 0^mdG3'5'p f o r methods #2-#4 was determined i n the same manner. I n experiments u s i n g method #2 without DNA the recovery was 39% and 37% when 1.13 and 1.69 X 1 0 " 1 4 moles 06mdG3'p was analysed. For d u p l i c a t e s of 1 pg dig e s t e d C3H10T1/2 DNA spiked w i t h 5.61 X 1 0 " 1 4 moles marker 0^mdG3'p and analysed u s i n g method #3, the recovery was 85%. Adding a second HPLC p u r i f i c a t i o n before the f i n a l chromatography (method #4) dropped the recovery to 51%. For a l l experiments, adduct l e v e l s were c o r r e c t e d f o r recovery, u s i n g i n t e r n a l c o n t r o l s or ' h i s t o r i c a l ' recovery values. 64 3.4 S e n s i t i v i t y 39 ft The s e n s i t i v i t y of the P - p o s t l a b e l i n g assay f o r 0 -methylguanine may be estimated as twice the background measured f o r a c o n t r o l DNA sample. I n t h i s context, ' c o n t r o l ' DNA i s DNA i s o l a t e d from c u l t u r e d c e l l s or t i s s u e s not exposed to meth y l a t i n g agents and th e r e f o r e assumed to c o n t a i n no 'exogenous' 0^-methylguanine. I h f i v e separate experiments DNA i s o l a t e d from C3H10T1/2 c e l l s and CHO c e l l s was analysed u s i n g method #1 under i d e n t i c a l c o n d i t i o n s and the estimated s e n s i t i v i t y , c o r r e c t e d f o r recovery, determined (Table 3). The average s e n s i t i v i t y i s 6.5 + 1.5 /zmole/mole. To confirm the s e n s i t i v i t y estimated from blank values of c o n t r o l DNA, samples of di g e s t e d DNA were spiked w i t h known amounts of s y n t h e t i c marker 0^mdG3'p and analysed. In a t y p i c a l experiment, 1 /ig samples of dig e s t e d C3H10T1/2 DNA were spiked w i t h s y n t h e t i c 06mdG3'p to simulate l e v e l s of 0 6-methylguanine i n DNA at 0.1, 0.7, 1.4,and 6.8 pmole/mole. V i s u a l examination of the r e s u l t i n g autoradiograms ( F i g 22) show th a t 0^mdG3'p i n dig e s t e d DNA to a l e v e l of 6.8 umol/mol i s c l e a r l y d e t e c t a b l e above background. In c o n t r a s t , the autoradiograms of DNA spiked w i t h 1.4 umol/mole 0^mdG3'p or lower are i n d i s t i n g u i s h a b l e from those of c o n t r o l (unspiked) DNA. The estimated s e n s i t v i t y of method #2 f o r a n a l y s i s of C3H10T1/2 DNA was 0.5 /zmole/mole. The a c t u a l s e n s i t i v i t y was assessed u s i n g DNA samples spiked w i t h 0 mdG3'p as desc r i b e d above. Calf-thymus DNA spiked w i t h 0.2 /xmole/mole 0^mdG3'p on a n a l y s i s gave a 0^mdG3'5'p spot and was v i s u a l l y d i s t i n g u i s h a b l e from the c o n t r o l sample ( F i g 23). g The estimated s e n s i t i v i t y (twice background) f o r d e t e c t i o n of 0 -methylguanine i n 1 /*g C3H10T1/2 DNA analysed u s i n g methods #3 and #4 was 8.5 and 2 /^mole/mole r e s p e c t i v e l y . 65 TABLE 3 SENSITIVITY OF THE 3 2P-POSTLABELING ASSAY FOR 06-METHYLGUANINE-METHOD #1 DNA Source ( c e l l type) S e n s i t i v i t y (umole/mole) C3H 8. .4 C3H 5. , l a C3H 5. ,2 a C3H 6. .1 CHO 7, .6 mean = 6 .5 Note: I n f i v e separate experiments, independent i s o l a t e s of 1 pg DNA from untreated CHO and C3H10T1/2 c u l t u r e s were analysed f o r 0 -methylguanine us i n g method #1. The s e n s i t i v i t y was estimated as twice the molar eq u i v a l e n t of the r a d i o a c t i v i t y i n the 0 mdG3'5'p zone of the chromatogram. Values were c o r r e c t e d f o r an adduct recovery of 84%. a Mean of d u p l i c a t e analyses. Figure 22 S e n s i t i v i t y of the J Z P - p o s t l a b e l i n g assay f o r 0 6-methylguanine-method #1 ( s i n g l e HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) . Digested samples of 1 ;ig DNA were spiked w i t h the i n d i c a t e d e q u i v a l e n t l e v e l s (umole/mole) of s y n t h e t i c 06mdG3'p and then assayed. B. Column blank r e s u l t i n g from the l a b e l i n g of 500 u l HPLC solvent c o l l e c t e d from the column before i n j e c t i o n of DNA samples. 67 B • t • j h 0.002 . m I • y r °-02 is T • m J NoP 1 [r #• • f ~ . . Figure 23 S e n s i t i v i t y of the P - p o s t l a b e l i n g assay f o r 0 -methylguanine-method #2 (dual HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) . Digested samples of 1 ,ug calf-thymus DNA were spiked w i t h the i n d i c a t e d l e v e l s (umole/mole) of 0 mdG3'p then assayed. B. Column blank. PC. P o s i t i v e c o n t r o l . 1 X 1 0 " 1 5 moles s y n t h e t i c 06mdG3'p i n j e c t e d onto the column a f t e r DNA i n j e c t i o n s . 68 DETECTION AND QUANTITATION OF O6-METHYLGUANINE IN THE DNA OF TISSUE-CULTURE CELLS TREATED WITH N-METHYL-N-NlTROSOUREA I n t r o d u c t i o n N-Methyl-N-nitrosourea (MNU), a model d i r e c t - a c t i n g carcinogen (IARC 1978), induces the formation of 0^-methylguanine adducts i n DNA both i n v i t r o and i n v i v o (Beranek et a l . 1980). This has been e s t a b l i s h e d p r i m a r i l y through treatment of c e l l s w i t h r a d i o l a b e l e d MNU, f o l l o w e d by i s o l a t i o n of the DNA, a c i d i c d e p u r i n a t i o n and s c i n t i l l a t i o n counting of l a b e l e d 0 -methylguanine a f t e r HPLC i s o l a t i o n . The l a b e l e d purine has been i d e n t i f i e d by c o - e l u t i o n w i t h a u t h e n t i c marker (Lutz 1979). Treatment of Chinese Hamster Ovary (CHO) c e l l s w i t h MNU r e s u l t s i n the formation of 0^-methylguanine residues (Morris et a l . 1983, Beranek et a l . 1983). Most CHO c e l l l i n e s are unable to e x c i s e / r e p a i r t h i s adduct (Goth-G o l d s t e i n 1980). C3H10T1/2 c e l l s , i n c o n t r a s t , possess a l k y 1 t r a n s f e r a s e a c t i v i t y and t h e r e f o r e are capable of r e p a i r . Nevertheless, 0^-methylguanine has been detected and q u a n t i t a t e d i n C3H10T1/2 c e l l s exposed to MNU (Topal and Baker 1982). 32 6 As a t r i a l of the newly developed P - p o s t l a b e l i n g assays f o r 0 -methylguanine, DNA from both r e p a i r d e f i c i e n t CHO c e l l s and r e p a i r p r o f i c i e n t C3H10T1/2 c e l l s t r e a t e d w i t h MNU was analysed f o r the presence of 06mdG3'5'p usi n g methods #1 and #2. 69 1. D e t e c t i o n and Q u a n t i t a t i o n of 0 -methylguanine i n the DNA of CHO C e l l s  Treated w i t h N-Methvl-N-nitrosourea Methods CHO c e l l s were seeded i n t o f l a s k s c o n t a i n i n g 50 ml MEM supplemented w i t h a n t i b i o t i c s and 10% f e t a l c a l f serum at a d e n s i t y of 0.5 X 10^ c e l l s per f l a s k . The c e l l s grew to confluence (2 X 10^ c e l l s per f l a s k ) a f t e r two days i n c u b a t i o n at 37°C i n a h u m i d i f i e d atmosphere c o n t a i n i n g 5% CO2. 10 f l a s k s c o n t a i n i n g c o n f l u e n t CHO c e l l c u l t u r e s were t r e a t e d w i t h MNU (0 to 19.3 mM). F i r s t , growth medium was poured o f f the c e l l s , then a pre-weighed amount of MNU was q u i c k l y d i s s o l v e d i n 50 ml 'wash' MEM and the r e s u l t i n g s o l u t i o n immediately added to the f l a s k , to cover the c e l l s . For the c o n t r o l f l a s k , 50 ml of 'wash' MEM (without MNU) was added to the c e l l s . Exposure of the c e l l s to MNU was f o r 3 hours at 37°C i n a h u m i d i f i e d incubator fed 5% C 0 2 and 95% a i r . A f t e r treatment, the medium c o n t a i n i n g MNU was removed from each f l a s k and the monolayer r i n s e d f o r a few seconds w i t h 10 ml of 0.1% t r y p s i n . The r i n s e was d i s c a r d e d then the monolayer detached by t r y p s i n i z a t i o n f o r 5 minutes at room temperature. The r e s u l t i n g c e l l suspension was then t r a n s f e r r e d to a 17 X 100 mm c e n t r i f u g e tube. The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n f o r 5 minutes at 2000 rpm. The supernatant was poured o f f then 10 ml phosphate b u f f e r e d s a l i n e (PBS) added and the c e l l s r e c e n t r i f u g e d to remove the l a s t t r a c e s of t r y p s i n . Once the PBS r i n s e was removed the c e l l s were d i s p e r s e d i n 1 ml f r e s h PBS, t r a n s f e r r e d to a 1.5 ml polypropylene tube and s t o r e d at -70°C. DNA was e x t r a c t e d from CHO c e l l s according to the method described on page 43. 70 The l e v e l of 0 6-methylguanine i n the DNA of CHO c e l l s t r e a t e d w i t h MNU was measured u s i n g method #1. Preceding the a n a l y s i s of dig e s t e d DNA samples, a 'column blank' c o n s i s t i n g of 0.5 ml HPLC sol v e n t through the column was c o l l e c t e d . Digested CHO DNA from the c o n t r o l sample was i n j e c t e d onto the HPLC f i r s t f o l l o w e d by the treatment samples i n order of exposure to i n c r e a s i n g concentrations of MNU. A f t e r i n j e c t i o n and c o l l e c t i o n of the "1 A C c o n t r o l DNA, a p o s i t i v e c o n t r o l sample c o n s i s t i n g of 2 X 10" moles 0 mdG3'p was i n j e c t e d onto the column and the appropriate f r a c t i o n c o l l e c t e d . R e s u l t s In the case of the two hi g h e s t doses of MNU used (9.6 and 19.3 mM), the medium pH was 6 at the end of the exposure p e r i o d , compared to 7.4 before treatment. The pH f o r the other doses remained unchanged compared to c o n t r o l . A l s o , most of the monolayer detached during treatment f o r the two hi g h e s t doses and as a r e s u l t very few c e l l s were recovered. These samples were t h e r e f o r e , not analysed. The average (based on U.V.) y i e l d of DNA e x t r a c t e d from one f l a s k of CHO c e l l s was 15 /ig. Autoradiograms of the column blank, 0^mdG3'p p o s i t i v e c o n t r o l and CHO DNA samples are shown i n Figure 24. The f i l m s of DNA from MNU t r e a t e d CHO c e l l s show a spot t h a t co-chromatographs w i t h 0 mdG3'5'p i n 2-dimensional TLC by comparison w i t h autoradiograms of the p o s i t i v e c o n t r o l . The spot i n t e n s i t y increases w i t h h i g her doses of MNU. From the r a d i o a c t i v i t y of the adduct 39 6 spots and us i n g the s p e c i f i c a c t i v i t y of P-ATP the l e v e l of 0 -methylguanine i n the CHO DNA was determined. The q u a n t i t a t i v e data are presented i n Figure 25. 71 Figure 24 D e t e c t i o n and q u a n t i t a t i o n of 0 -methylguanine i n CHO c e l l s t r e a t e d w i t h MNU. Autoradiograms show the r e s u l t s of exposing c e l l s to the i n d i c a t e d concentrations (mM) of MNU and then a n a l y s i n g the DNA usin g method #1 ( s i n g l e HPLC p u r i f i c a t i o n -diphosphate l e v e l ) . B. Column blank c o n s i s t i n g of 500 >il HPLC s o l v e n t from the column before DNA i n j e c t i o n s . PC. P o s i t i v e c o n t r o l - 2 X 1 0 " 1 4 moles s y n t h e t i c 06mdG3'p i n j e c t e d onto the column a f t e r DNA i n j e c t i o n s . C i r c l e d area- 0 mdG3'5'p spot v i s i b l e on o r i g i n a l autoradiogram. 72 • B 1 .origin • i t • 0.08 •o • • 0 • • .0.34 | 0.58 . . ' t 122 «# •••• • "ST • 4.82 < • PC M A ' • % • Figure 24 73 I 1 1 1 1 1 1 1 1 1 0.50 1.00 2.00 3.00 4.00 5.00 Methylnitrosourea (mM) Figure 25 Dose-response of 0-methylguanine i n the DNA of CHO c e l l s t r e a t e d w i t h MNU. 74 2. D e t e c t i o n and Q u a n t i t a t i o n of 0 6-methylguanine i n the DNA of C3H10T1/2  Mouse Embryo F i b r o b l a s t C e l l s t r e a t e d w i t h N-Methvl-N-nitrosourea Methods Mouse embryo f i b r o b l a s t c e l l s (passage 13) were seeded i n t o 16 f l a s k s (each c o n t a i n i n g 20 ml BME supplemented w i t h 10% f e t a l c a l f serum and a n t i b i o t i c s ) at a d e n s i t y of 0.5 X 10^ c e l l s per f l a s k . A f t e r four days i n c u b a t i o n at 37°C v i s u a l i n s p e c t i o n r e v e a l e d that the c e l l s had grown to confluence. E x c l u d i n g c o n t r o l (no chemical) f l a s k s , the f r e s h l y c o n f l u e n t C3H10T1/2 c e l l s were exposed i n d u p l i c a t e to 7 d i f f e r e n t concentrations of MNU. For a given dose of MNU, medium was removed from the f l a s k s then 20 ml of medium c o n t a i n i n g f r e s h l y d i s s o l v e d MNU was added to each f l a s k . Treatment was f o r 3 hours at 37^C i n a h u m i d i f i e d incubator fed 5% CC>2 and 95% a i r . C e l l s were harvested by t r y p s i n i z a t i o n according to a m o d i f i c a t i o n of the procedure des c r i b e d p r e v i o u s l y f o r CHO c e l l s . DNA from C3H10T1/2 c e l l s exposed to MNU was analysed f o r O^-methylguanine u s i n g method #1. I n r e l a t i o n to c e l l l i n e s which are incapable of r e p a i r i n g t h i s adduct (e.g. CHO c e l l s ) the l e v e l of 0 6-methylguanine i n C3H10T1/2 c e l l s t r e a t e d w i t h an e q u i v a l e n t dose of MNU would be expected to be s i g n i f i c a n t l y lower. To increase the chances of d e t e c t i n g 0^-methylguanine i n C3H10T1/2 c e l l s , 10% of the d i l u t e d l a b e l e d kinase r e a c t i o n was f u r t h e r p u r i f i e d by reverse-phase HPLC, according to method #2. A column blank c o n s i s t i n g of 700 ]x\ HPLC s o l v e n t passed through the column was obtained before a c t u a l sample i n j e c t i o n s . S c i n t i l l a t i o n counting was done only f o r samples analysed using method #2. 75 The s u r v i v a l of C3H10T1/2 c e l l s exposed to MNU was determined by a colony forming assay ( R e z n i k o f f e t a l . 1973). 200 c e l l s were seeded i n t o each of 24-60 mm counting dishes c o n t a i n i n g 5 ml BME + 10% FCS and a n t i b i o t i c s . This was done at the same time and w i t h the same c e l l s used f o r 0^-methylguanine a n a l y s i s (see above). One day a f t e r seeding, the c e l l s were exposed i n t r i p l i c a t e (3 dishes/dose MNU) to the same concentrations of MNU used f o r the p o s t l a b e l i n g a n a l y s i s (see above). For each l e v e l of MNU, the medium was removed from 3 dishes then 5 ml medium c o n t a i n i n g f r e s h l y d i s s o l v e d MNU was added to each d i s h . I ncubation was f o r 3 hours a t 37°C. A f t e r treatment the chemical was rep l a c e d w i t h 5 ml f r e s h 'growth' medium and the c e l l s allowed to grow f o r a f u r t h e r 8 days (a medium change was made on the f i f t h day). The s u r v i v a l of C3H10T1/2 c e l l s exposed to MNU i n terms of t h e i r colony forming c a p a c i t y was determined by comparing the number of c o l o n i e s i n dishes t r e a t e d w i t h MNU to those i n the c o n t r o l dishes (without MNU). In p r e p a r a t i o n f o r colony counting, the dishes were r i n s e d twice i n d i s t i l l e d water, f i x e d i n 95% e t h a n o l , a i r d r i e d and the c o l o n i e s were v i s u a l i z e d by s t a i n i n g w i t h 0.1% methylene blue. Colonies c o n t a i n i n g 20 or more c e l l s were i n c l u d e d i n the a n a l y s i s . R e s u l t s The average y i e l d of DNA i s o l a t e d from two conflu e n t f l a s k s was 60.7 /ig (based on U.V. a b s o r p t i o n ) . Autoradiograms of C3H10T1/2 DNA analysed using method #1 are shown i n Figure 26, A spot t h a t co-chromatographs w i t h l a b e l e d s y n t h e t i c 0^mdG3'5'p i s present f o r MNU concentrations of 1.27 mM and higher. Maps of DNA, analysed u s i n g method #2 are shown i n Figure 27. Adduct spots are present f o r a l l concentrations of MNU used. An MNU/0 -methylguanine dose-76 Figure 26 D e t e c t i o n and q u a n t i t a t i o n of 0 -methylguanine i n the DNA of C3H10T1/2 c e l l s t r e a t e d w i t h MNU. Shown, are autoradiograms r e s u l t i n g from the exposure of c e l l s to the i n d i c a t e d dose (mM) of MNU f o l l o w e d by p o s t l a b e l i n g a n a l y s i s of DNA u s i n g method #1 ( s i n g l e HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) . B. Column blank. PC. P o s i t i v e c o n t r o l c o n s i s t i n g of 2 X 10 moles s y n t h e t i c 0 mdG3'p i n j e c t e d onto the column a f t e r DNA i n j e c t i o n s . 77 B f 1 I ,origin 0 - - ' 0.08 ^ ^ ^ f ,t ^ ^ ^ ^ ^ ^ * • 0.15 ^ | f • • 0.26 m • ! • 1.27 - n A* • 2.48 ^ • f i 4.97 » * T PC ' Figure 26 78 Figure 27 Autoradiograms of DNA from C3H10T1/2 c e l l s exposed to va r i o u s doses (mM) of MNU. DNA was analysed by P - p o s t l a b e l i n g using method #2 (dual HPLC p u r i f i c a t i o n - d i p h o s p h a t e l e v e l ) . B. Column blank. PC. P o s i t i v e c o n t r o l - 2 X 1 0 " ^ moles s y n t h e t i c 06mdG3'p i n j e c t e d onto the column a f t e r DNA i n j e c t i o n s . 79 B .origin / • 0.15 m * \ • i t i # • 0.26 # » \ . V 0.57 « • ( / " 1.27 % +\ 2.48 « • \ M B 4.97 f | if PC + • • • Figure 27 80 response curve i l l u s t r a t i n g adduct l e v e l s determined u s i n g method #2 i s shown i n Figure 28. The autoradiograms from a n a l y s i s of C3H10T1/2 DNA (Figure 26 ) u s i n g method #1 show many a d d i t i o n a l background spots compared to autoradiograms of CHO DNA (Figure 24), i n c l u d i n g one that p a r t i a l l y i n t e r f e r e s w i t h 06mdG3'5'p. Using an HPLC method, these spots were t r a c e d to a markedly higher RNA content f o r the C3H10T1/2 samples and are presumably r e s i d u a l r i b o n u c l e o s i d e 3'5'-bisphosphates. A d d i t i o n a l HPLC p u r i f i c a t i o n of l a b e l e d 0^mdG3'5'p l a r g e l y removed t h i s p u t a t i v e RNA a s s o c i a t e d spot, reducing i n t e r f e r e n c e and improving the s e n s i t i v i t y of q u a n t i t a t i o n . RNA contamination of DNA samples can a l s o be e l i m i n a t e d by repeated ethoxyethanol p r e c i p i t a t i o n s (data not shown). R e s u l t s of the assay f o r c e l l s u r v i v a l are shown i n Figure 29. The p l a t i n g e f f i c i e n c y of these c e l l s was 25%. These data show th a t MNU i s very t o x i c toward C3H10T1/2 c e l l s . From Figure 28 and i n t e r p o l a t i o n of the curve i n Figure 29, the l e v e l of O^-methylguanine a s s o c i a t e d w i t h 50% s u r v i v a l was 3.5 pmole/mole. 81 100.CH 0.1 H 1 1 1 1 1 0 1.00 3.00 5.00 Methylnitrosourea (mM) Figure 28 Dose-response of 0 -methylguanine i n the DNA of C3H10T1/2 mouse-embryo f i b r o b l a s t c e l l s t r e a t e d w i t h MNU. For each dose of MNU, DNA was analysed f o r 0 -methylguanine by P - p o s t l a b e l i n g using method #2. 82 100.0 50.0-- 20.0 c o o CD > CD CO > 3 CO 10.0-Methy ln i t rosourea (mM) Figure 29 S u r v i v a l of C3H10T1/2 mouse-embryo f i b r o b l a s t c e l l s exposed to MNU. 200 c e l l s were seeded i n t o dishes and the next day, exposed to MNU f o r 3 hours at 37°C. A f t e r treatment, the medium was replac e d and the c e l l s incubated at 37°C f o r a f u r t h e r 8 days. Each data p o i n t represents an average of three p l a t e s per dose. 83 DISCUSSION AND CONCLUSIONS 32 The primary goals of t h i s p r o j e c t were to develop a s e n s i t i v e P-p o s t l a b e l i n g assay f o r 0^-methylguanine r e q u i r i n g only microgram q u a n t i t i e s of DNA and to assess the f e a s i b i l i t y of i t s a p p l i c a t i o n to adduct d e t e c t i o n i n humans. 1. Method Development 0°mdG3'p, r e q u i r e d as a chromatography marker f o r development of the method, was prepared u s i n g a novel two-stage approach. S i m i l a r i t y of the U.V. spectrum of 0 mdG3'p to that of 0 mdG i n d i c a t e s r e t e n t i o n of the 0 -methylated guanine base. Conversion to O^ mdG by nuclease PI and r e s i s t a n c e to h y d r o l y s i s by 5'-nucleotidase i s c o n f i r m a t i o n t h a t the compound i s a deoxynucleoside 3'-monophosphate. This i s the f i r s t r eported s y n t h e s i s of the mod i f i e d n u c l e o t i d e . There are syntheses a v a i l a b l e f o r the deoxynucleoside form of many DNA adducts. The authe n t i c standards necessary f o r the development of p o s t l a b e l i n g assays f o r s e v e r a l other a l k y l a t e d DNA adducts such as 0^-methylthymine might t h e r e f o r e be synth e s i z e d by t h i s route. The a b i l i t y of 06mdG3'p to be 3 2 P - l a b e l e d by p o l y n u c l e o t i d e kinase and 3 2P-ATP i s f u r t h e r c o n f i r m a t i o n of the compound's i d e n t i t y and i s the b a s i s f o r a method to measure 0^-methylguanine i n DNA by p o s t l a b e l i n g . Reverse-phase HPLC was used to i s o l a t e 0^mdG3'p from normal n u c l e o t i d e s 32 before p o s t l a b e l i n g . HPLC p u r i f i c a t i o n reduces the amount of P-ATP req u i r e d f o r q u a n t i t a t i v e l a b e l i n g of 0^mdG3'p and the a s s o c i a t e d l e v e l of r a d i o a c t i v i t y i n the assay. 0^mdG3'p i s s u s c e p t i b l e to enzymatic d i g e s t i o n by nuclease P I , t h e r e f o r e enrichment procedures based on s e l e c t i v e dephosphorylation of normal n u c l e o t i d e s are not a p p l i c a b l e (Reddy and Randerath 1986) . HPLC enrichment r e s u l t e d i n an approximately 1000 f o l d 84 r e d u c t i o n i n the amount of normal n u c l e o t i d e s r e l a t i v e to 0^mdG3'p (data not shown). HPLC chromatography of DNA d i g e s t s enabled the exact amount of dige s t e d normal n u c l e o t i d e s a s s o c i a t e d w i t h a kinase r e a c t i o n to be determined. This should improve the accuracy si n c e assumptions based on U.V. qu a n t i t a t e d DNA before d i g e s t i o n are e l i m i n a t e d . The recovery and a n a l y s i s of s e v e r a l d i f f e r e n t adducts i n one chromatographic run i s p o s s i b l e i f the corresponding markers are a v a i l a b l e . A f t e r p o s t l a b e l i n g of the HPLC f r a c t i o n c o n t a i n i n g 06mdG3'p, 06mdG3'5'p was r e s o l v e d from r e s i d u a l l a b e l e d normal n u c l e o t i d e s u s i n g two-dimensional P E I - c e l l u l o s e t h i n - l a y e r chromatography; t h i s i s necessary f o r q u a n t i t a t i o n . This embodies the b a s i c procedure (method #1) f o r a n a l y i s of 0^-methylguanine 32 by P - p o s t l a b e l i n g . The procedural steps of t h i s method and three v a r i a t i o n s (methods #2-#4) i n v e s t i g a t e d to improve the s e n s i t i v i t y are summarized i n Figure 16. 2. Comparison of Methods The performance c h a r a c t e r i s t i c s of the four methods are given i n Table 4. HPLC p u r i f i c a t i o n of 0^mdG3'5'p (method #2) lowered the background enabling the d e t e c t i o n of ten times l e s s adduct compared to method #1 even though the recovery dropped s i g n i f i c a n t l y to 38%. The improved performance i s at the expense of exposure to more r a d i o a c t i v i t y and a s l i g h t l y longer a n a l y s i s time. I t has been reported t h a t conversion of l a b e l e d normal n u c l e o t i d e s and adduct n u c l e o t i d e s , r e s u l t i n g from the a n a l y s i s of DNA t r e a t e d w i t h methylating agents, to t h e i r r e s p e c t i v e 5'-monophosphates reduces the background i n the adduct area of the TLC chromatograms (Reddy e t a l . 1984). This prompted the development of methods #3 and #4 based on treatment of the kinase r e a c t i o n w i t h nuclease PI. In c o n t r a s t to the l i t e r a t u r e evidence, the r e s u l t s show that a n a l y s i s at the 5'-monophosphate l e v e l d i d not improve the s e n s i t i v i t y . 85 TABLE 4 PERFORMANCE CHARACTERISTICS OF THE 3 2P-P0STLABELING ASSAYS FOR O6-METHYLGUANINE Method 1 2 3 4 Time 1 1 (days) 5 6 9 10 R a d i a t i o n low h i g h low hig h S e n s i t i v i t y (pmole/mole) 6.5 + 1.5 0.5 8.5 2.0 Recovery (%) 84 38 85 51 a Time of a n a l y s i s from t i s s u e to r e s u l t , k Approximate operator exposure. ° Estimated s e n s i t i v i t y d e f i n e d as twice the background r a d i o a c t i v i t y i n the adduct zone f o r a n a l y s i s of 1 pg DNA from untreated t i s s u e - c u l t u r e c e l l s . The value f o r method #1 was e x t r a c t e d from Table 1. The r e s t of the values are from a s i n g l e experiment and are the average of d u p l i c a t e analyses. ^ HPLC a n a l y s i s of 1 pg DNA spiked w i t h standard 0^mdG3'p compared to the same amount of standard 0^mdG3'p l a b e l e d d i r e c t l y . The data f o r methods #1 and #2 are averages from two experiments. 86 Methods #3 and #4 are much longer, t e c h n i c a l l y more d i f f i c u l t and expose the operator to more r a d i a t i o n compared to methods #1 and #2. In co n c l u s i o n , t h i s work shows th a t methods #1 and #2 are s u p e r i o r to methods #3 and #4 and that p u r i f i c a t i o n of 06mdG3'5'p by HPLC (method #2) allows d e t e c t i o n of 0.5 micromole 0^-methylguanine per mole of normal n u c l e o t i d e i n 1 microgram of DNA. 32 3. Factors L i m i t i n g the Performance of the P - P o s t l a b e l i n g Assays 3.1 R e p r o d u c i b i l i t y The standard d e v i a t i o n f o r r e p l i c a t e a n a l y s i s (method #1) of s y n t h e t i c 06mdG3'p i s + 6% (Table 1). This i s evidence t h a t the r e p r o d u c i b i l i t y of the assay i s h i g h f o r repeated samplings from a s i n g l e DNA d i g e s t . The major ' w i t h i n d i g e s t ' v a r i a t i o n probably a r i s e s from e r r o r s i n c u t t i n g out the 0^mdG3'5'p spot along w i t h infringement of the adduct zone by background spots. The r e p r o d u c i b i l i t y of DNA d i g e s t i o n determined by separate i n j e c t i o n and HPLC a n a l y s i s of DNA a l i q u o t s taken from a s i n g l e s o l u t i o n i s higher w i t h i n an experiment than between (Table 2). The higher between experiment v a r i a t i o n i n d i g e s t i o n c o u l d a r i s e from e r r o r s i n U.V. q u a n t i t a t i o n of DNA. V a r i a b l e RNA contamination of samples co u l d c o n t r i b u t e because HPLC q u a n t i t a t i o n i s s p e c i f i c f o r DNA n u c l e o t i d e s . These data suggest t h a t the re l e a s e of 0^mdG3'p from DNA i s re p r o d u c i b l e and tha t m u l t i p l e a n a l y i s of s i n g l e DNA s o l u t i o n s are not necessary. The assay r e p r o d u c i b l i t y as determined from m u l t i p l e a n a l y s i s of one DNA sample should be higher than the r e p r o d u c i b l i t y of DNA d i g e s t i o n determined here. This i s because the adduct l e v e l s (what the assay measures) are expressed as a r a t i o of moles of adduct to the d i g e s t i o n y i e l d f o r each i n d i v i d u a l i n j e c t i o n and t h i s r a t i o should be independent of d i g e s t i o n . The r e p r o d u c i b i l i t y of the assay may be reduced at 87 low adduct l e v e l s because the background forms a l a r g e r percentage of the r a d i o a c t i v i t y measured i n the 0^mdG3'5'p zone. 3.2 Recovery The recovery of 0^mdG3'p a f t e r enzymatic r e l e a s e from DNA was about 85% f o r methods #1 and #3 (Table 4 ) . Further HPLC p u r i f i c a t i o n of the l a b e l e d adduct (methods #2 and #4) reduced the recovery to below 50%. The adduct r e c o v e r i e s determined here f o r 0^mdG3'p are q u i t e c o n s i s t e n t w i t h those found f o r other adducts measured u s i n g p o s t l a b e l i n g methodology. Recoveries of l a b e l e d adducts were 40-50% and 70% f o r DNA mo d i f i e d i n v i t r o w i t h N-hydroxy-2-aminofluorene and a di o l - e p o x i d e of benzo[a]pyrene r e s p e c t i v e l y (Gupta et a l . 1982). P o s t l a b e l i n g a n a l y s i s of DNA mo d i f i e d i n v i t r o w i t h 2-acetylaminofluorene, b e n z i d i n e , and 4-dimethylaminoazobenzene gave adduct r e c o v e r i e s of 45-50% (Reddy e t a l . 1984). F i n a l l y , a n a l y s i s by 32 P - p o s t l a b e l i n g of def i n e d mixtures of normal deoxynucleoside 3'-2 monophosphates and s y n t h e t i c N -(dehydro-estragol-1'-yl)-deoxyguanosine-3'-phosphate showed th a t recovery of the l a b e l e d adduct was about 60% tha t of the normal n u c l e o t i d e s ( F e n n e l l e t a l . 1986). There are two mechanisms by which 0^mdG3'p cou l d be l o s t during the process of chromatography and l a b e l i n g . These " s i n k s ' are e i t h e r chemical/enzymatic or p h y s i c a l . Under the f i r s t mentioned category, decomposition of the adduct n u c l e o t i d e s i n low pH (3.5) ammonium formate during HPLC and TLC may be important. O^ mdG i s about 50 times l e s s s t a b l e to de p u r i n a t i o n than the unmethylated deoxynucleoside i n a c i d i c c o n d i t i o n s (Farmer et a l . 1973). Losses i n c u r r e d when a second HPLC p u r i f i c a t i o n i s performed may i n p a r t be due to simply a longer contact w i t h low pH solvent . Drying down the adduct n u c l e o t i d e s may te m p o r a r i l y expose them to a combination of h i g h s t r e n g t h ammonium formate/low pH and a c c e l e r a t e the 88 decomposition. P h y s i c a l l o s s e s may occur through i r r e v e r s i b l e absorption. This can happen during HPLC i t s e l f or through incomplete uptake of the d r i e d down f r a c t i o n from the tube before l a b e l i n g . Using a r a d i a t i o n d e t e c t o r , i t was apparent t h a t not a l l of the r a d i o a c t i v i t y was recovered from the tube a f t e r a d d i t i o n of the HPLC c a r r i e r preparatory to the second p u r i f i c a t i o n (data not shown). 3.3 S e n s i t i v i t y The s e n s i t i v i t y of the p o s t l a b e l i n g technique f o r DNA adduct d e t e c t i o n i s enhanced when the t a r g e t adducts are separable as a c l a s s from the normal n u c l e o t i d e s before p o s t l a b e l i n g . L i m i t s of d e t e c t i o n f o r aromatic and/or bulky adducts are very low (0.0001 to 0.001 ^ mole/mole) because i n these cases normal n u c l e o t i d e s are e f f e c t i v e l y e l i m i n a t e d by e x t r a c t i o n , chromatographic i s o l a t i o n or enzymatic d i g e s t i o n p r i o r to l a b e l i n g . 0^mdG3'p d i f f e r s by only a methyl group from unmodified n u c l e o t i d e s and th e r e f o r e i s d i f f i c u l t to re s o l v e from them. Development of a s e n s i t i v e p o s t l a b e l i n g method to detect t h i s adduct represents a s i g n i f i c a n t challenge to the chromatographer. I t i s apparent from t h i s work th a t the s e n s i t i v i t y of the method i s not hampered by an i n a b i l i t y to detect 0^mdG3'p but r a t h e r by the background. This can be demonstrated by, f o r example assuming that an adduct l e v e l g i v i n g 32 100 dpm above background i s measurable w i t h some r e l i a b i l i t y . I f the P-ATP -18 s p e c i f i c a c t i v i t y i s 3000 Ci/mmole then t h i s corresponds to 7.51 X 10" moles adduct. Assuming the adduct arose from a n a l y s i s of 1 /ig DNA or about 3 nmole DNA-p then the l e v e l of adduct i n the DNA would be 0.005 pmole/mole thereby d e f i n i n g the p o s s i b l e s e n s i t i v i t y i n the absence of chromatographic background. 89 HPLC i s o l a t i o n of 0^mdG3'p from e n z y m a t i c a l l y d i g e s t e d DNA does not remove a l l normal n u c l e o t i d e s and compounds which are l a b e l e d by P-ATP and p o l y n u c l e o t i d e k i n a s e . Some of t h i s l a b e l e d r e s i d u a l m a t e r i a l e l u t e s w i t h and very c l o s e to 0^mdG3'5'p i n the two dimensional TLC system. These i m p u r i t i e s t h e r e f o r e , reduce the s e n s i t i v i t y and r e p r o d u c i b i l i t y of the assay. The major c o n t r i b u t i o n s to the background seen i n c o n t r o l samples are a) n o n - s p e c i f i c 32 r a d i o a c t i v i t y from P-ATP and o l i g o n u c l e o t i d e s , b) assay and RNA r e l a t e d background spots and c) t a i l i n g r a d i o a c t i v i t y from r e s i d u a l dA3'5'p. An a d d i t i o n a l p u r i f i c a t i o n stage before PEI-TLC reduced these i m p u r i t i e s (method #2). This was accomplished by chromatography of the l a b e l e d kinase mixture on a reverse-phase column f o l l o w e d by c o l l e c t i o n of the 0^mdG3'5'p f r a c t i o n . The t h e o r e t i c a l s e n s i t i v i t y of the p o s t l a b e l i n g assay when a p p l i e d to human DNA w i l l depend on the 'ba s e l i n e ' l e v e l s of O^-methylguanine. Sources of t h i s adduct are ubiquitous N-nitrosamines i n the environment and non-enzymatic m e t h y l a t i o n of DNA by the i n t r a c e l l u l a r methyl group donor, S-adenosyl-L-methionine (Barrows and Shank 1981, Rydberg and L i n d a h l 1982). B a s e l i n e l e v e l s w i l l a l s o be i n f l u e n c e d by the a l k y l t r a n s f e r a s e a c t i v i t y of the p a r t i c u l a r t i s s u e . The s e n s i t i v i t y of the method (0.5 /xmole/mole) exceeds t h a t of techniques based on HPLC fluorescence or s c i n t i l l a t i o n counting of ^ 4C l a b e l e d purines a f t e r treatment w i t h l a b e l e d chemicals (Herron and Shank 1979, B a i r d 1979). I t performs about as w e l l as immunoassays and radioimmunoassays which t y p i c a l l y r e q u i r e l a r g e r (1000 pg) samples of DNA (Wild et a l . 1983, 32 ft Castonguay et a l . 1985). The P - p o s t l a b e l i n g assay f o r 0 -methylguanine should be more s e l e c t i v e than immunoassays f o r t h i s adduct because of the numerous chromatographic i s o l a t i o n s and p u r i f i c a t i o n s done before q u a n t i t a t i o n . 90 4. D e t e c t i o n and Q u a n t i t a t i o n of 0^-methylguanine i n Mammalian Tissue-Culture  C e l l s Treated w i t h N-Methyl-N-nitrosourea 3 2 P - P o s t l a b e l i n g a n a l y s i s of DNA from r e p a i r p r o f i c i e n t (C3H10T1/2) and r e p a i r d e f i c i e n t (CHO) mammalian t i s s u e - c u l t u r e c e l l s t r e a t e d w i t h MNU gives a ft ft compound th a t co-chromatographs w i t h s y n t h e t i c marker 0 mdG3'p or 0 mdG3'5'p i n four d i f f e r e n t systems (Figures 24, 26 and 27). This was not observed f o r untreated c u l t u r e s . Under s i m i l a r treatment c o n d i t i o n s , MNU has beeen shown to induce the formation of 0^-methylguanine i n these c e l l l i n e s u s i n g methods completely u n r e l a t e d to p o s t l a b e l i n g (Morris et a l . 1983, Beranek et a l . 1983, Topal and Baker 1982)). In the t r i a l w i t h C3H10T1/2 c e l l s , the lower background r e s u l t i n g from an a d d i t i o n a l HPLC p u r i f i c a t i o n enabled the d e t e c t i o n of adduct at a l l dosage l e v e l s , even t h a t a l l o w i n g 70% s u r v i v a l ( F i g 27). These data suggest a h i g h negative c o r r e l a t i o n ( c o e f f i c i e n t = -0.992) between the s u r v i v a l of C3H10T1/2 c e l l s and l e v e l s of 0^-methylguanine f o r low doses of MNU. This r e s u l t i s c o n s i s t e n t w i t h other evidence which suggests t h a t t h i s adduct p l a y s a c e n t r a l r o l e i n c e l l k i l l i n g by N-nitrosamines (Doniger et a l . 1985). 5. Recommendations to Enable A p p l i c a t i o n of the Method to Human Studies The l e v e l of 0^-methylguanine adducts i n the DNA of e x f o l i a t e d c e l l s from humans c h r o n i c a l l y exposed through h a b i t or environment to N-nitrosamines i s probably very low. There i s both d i r e c t and i n d i r e c t evidence that suggests t h i s may be the case. F i r s t , the c a p a c i t y of most human t i s s u e s s t u d i e d f o r r e p a i r of t h i s l e s i o n i s very h i g h (Grafstrom e t a l . 1984, H a l l et a l . 1985). Secondly, some t i s s u e s from people exposed to p u t a t i v e N-nitrosamines have been analysed f o r 0^-methylguanine u s i n g radioimmunoassays. In one r e p o r t , t h i s adduct was not detected i n the placentae of 10 women who smoked during 91 pregnancy, u s i n g a radioimmunoassay w i t h a s e n s i t i v i t y of 0.19 pmole/mole (Everson et a l . 1987). I n another r e p o r t , esophageal t i s s u e s from a p o p u l a t i o n a t an e l e v a t e d r i s k f o r esophageal cancer due to p o s s i b l e N-nitrosamine exposure from the d i e t were analysed f o r t h i s adduct (Umbenhauer et a l . 1985). The radioimmunoassay used i n t h i s study detected a h i g h value of 0.05 pmole/mole 0 -methylguanine i n one i n d i v i d u a l and lower values i n others when 1000 pg of DNA was employed. To be u s e f u l f o r the d e t e c t i o n of low 0 -methylguanine l e v e l s ( l e s s than 0.05 jumole/mole) i n small samples of DNA from e x f o l i a t e d human c e l l s , the s e n s i t i v i t y of the p o s t l a b e l i n g method must be increased by a f a c t o r of ten; t h i s should be the focus of f u t u r e method development. The r e q u i r e d improvement i n s e n s i t i v i t y c o u l d be achieved through a n a l y s i s of more DNA (up to ten micrograms) i n combination w i t h r e d u c t i o n of r a d i o a c t i v e background i n the adduct zone. Lowering of background might be e f f e c t e d by i n c r e a s i n g the e f f i c i e n c y of the chromatographic separations inherent i n the method. This i s p r e f e r r e d over i n c l u d i n g more HPLC p u r i f i c a t i o n steps which would cause f u r t h e r l o s s e s of adduct. Improving the r e s o l u t i o n of 0^mdG3'p from normal n u c l e o t i d e s i n the i n i t i a l HPLC step using d i f f e r e n t s o l v e n t systems perhaps i n combination w i t h other columns may lower the l e v e l of i m p u r i t i e s i n the adduct f r a c t i o n . HPLC separations u s i n g a so l v e n t of higher pH may a l s o improve the recovery of 0^mdG3'p. The accuracy of the P - p o s t l a b e l i n g assay f o r 0 -methylguanine should be d i r e c t l y v a l i d a t e d . Simultaneous measurement / a n a l y s i s of a DNA sample c o n t a i n i n g 0 -methylguanine us i n g the p o s t l a b e l i n g assay and another method would serve t h i s purpose. For example, 0^-(^C) -methylguanine forms i n the DNA of CHO c e l l s t r e a t e d w i t h 1 4 C - l a b e l e d MNU (Goth-Goldstein 1980). A f t e r DNA e x t r a c t i o n , some of the m a t e r i a l could be depurinated i n a c i d and i s o l a t e d from the h y d r o s y l a t e by HPLC u s i n g a u t h e n t i c 0 -methylguanine as a U.V. 92 marker. The l a b e l e d purine could then be q u a n t i t a t e d by s c i n t i l l a t i o n counting. Several other a l i q u o t s of the DNA co u l d be analysed by p o s t l a b e l i n g and the l e v e l s of adduct determined u s i n g the d i f f e r e n t methods d i r e c t l y compared. More data on the r e p r o d u c i b i l i t y of the method should be obtained. Of relevance i s the standard d e v i a t i o n of values from r e p l i c a t e a n a l y s i s of a l i q u o t s of a DNA sample c o n t a i n i n g 0^-methylguanine. The s e n s i t i v i t y of the method c o u l d be s u b s t a n t i a l l y improved i f the l e v e l of background were r e p r o d u c i b l e . The adduct l e v e l s could then be d e r i v e d by s u b t r a c t i o n of background r a d i a t i o n i n c o n t r o l samples from that of samples c o n t a i n i n g low l e v e l s of 0^-methylguanine. 93 REFERENCES Abbott, P.J., and S a f f h i l l , R. (1979) DNA synth e s i s w i t h methylated p o l y (dC-dG) templates. Evidence f o r a competitive nature to miscoding by 0^-methylguanine. Biochim. Biophys. Acta., 562, 51-61. Abbott, P.J., Metha, J.R., and Ludlum, D.B. (1980) Synthesis of 8- 1 4C- l a b e l e d 0 -methyldeoxyguanosine and i t s deoxynucleotide copolymers. Biochemistry, 19, 643-647. Arce, G.T., C l i n e , D.T.,Jr., and Mead, J.E. (1987) The 3 2 P - p o s t l a b e l i n g method i n q u a n t i t a t i v e DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity i n Chinese hamster ovary c e l l s and Salmonella typhimurium TA1538. Carcinogenesis, 8, 515-520. Autrup, H., and H a r r i s , C.C. Metabolism of chemical carcinogens by human t i s s u e s . In: H a r r i s , C.C, and Autrup, H. eds. Human Carcinogenesis. New York: Academic Press, 1983. pp 169-194. B a i r d , W.M. The use of r a d i o a c t i v e carcinogens to detect DNA m o d i f i c a t i o n s . In: Grover, P.L., ed. Chemical Carcinogens and DNA. Boca Raton, F l o r i d a : CRC Press. 1979, V o l . 1, pp 59-83. Barbacid, M. (1986) Oncogenes and human cancer: cause or consequence? Carcinogenesis, 7, 1037-1042. Barrows, R.L., and Shank, R.C. (1981) Aberrant m e t h y l a t i o n of l i v e r DNA i n r a t s d u r ing h e p a t o t o x i c i t y . T o x i c o l . Appl. Pharmacol., 60, 334-345. Bartch, H., and Montesano, R. (1984) Relevance of nitrosamines to human cancer. Carcinogenesis, 5, 1381-1393. Beranek, D.T. , Weis, C.C, and Swenson, D.H. (1980) A comprehensive q u a n t i t a t i v e ' a n a l y s i s of methylated and e t h y l a t e d DNA u s i n g h i g h pressure l i q u i d chromatography. Carcinogenesis, 1, 595. Beranek, D.T., H e f l i c h , R.H., K o d e l l , R.L., M o r r i s , S.M., and Casciano, D.A. (1983) C o r r e l a t i o n between s p e c i f i c DNA methylation products and mutation i n d u c t i o n a t the HGPRT locus i n Chinese hamster ovary c e l l s . Mutat. Res., 110, 171-180. Bernadou, J . , B l a n d i n , M., and Meunier, B. (1983) A one step s y n t h e s i s of 0 -methyldeoxyguanosine. Nucleosides and Nu c l e o t i d e s . , 2, 459-464. Bertram, J.S., K o l o n e l , L.N., and Meyskens, F.L. J r . (1987) R a t i o n a l e and s t r a t e g i e s f o r chemoprevention of cancer i n humans. Cancer Res., 47, 3012-3031. Bhanot, O.S., and Ray, A. (1986) The i n v i v o mutagenic frequency and s p e c i f i c i t y of 0^-methylguanine i n 0X174 r e p l i c a t i v e form DNA. Proc. N a t l . Acad. S c i . USA, 83, 7348-7352. Bishop, J.M. (1987) The molecular genetics of cancer. Science, 235, 305-311. 94 B o d e l l , W.J., and Rasmussen, J . (1984) A P - p o s t l a b e l i n g assay f o r determining the i n c o r p o r a t i o n of bromodeoxyuridine i n t o c e l l u l a r DNA. Anal. Biochem., 152, 275-284. Bohr, V.A., P h i l l i p s , D.H., and Harrawalt, P.C. (1987) Heterogeneous DNA damage and r e p a i r i n the mammalian genome. Cancer Res., 47, 6426-6436. Castonguay, A., F o i l e s , P.G., Trushin, N., and Hecht, S.S. (1985) Study of DNA me t h y l a t i o n by t o b a c c o - s p e c i f i c N-nitrosamines. Env. H e a l t h Perspect., 62, 197-202. Cayama, E., Tsuda, H., Sarma, D.S.R., and Faber, E. (1978) I n i t i a t i o n of chemical c a r c i n o g e n e s i s r e q u i r e s c e l l p r o l i f e r a t i o n . Nature, 275, 60-62. Chacko, M., and Gupta, R.C. (1987) E v a l u a t i o n of DNA damage i n the o r a l mucosa of c i g a r e t t e smokers and nonsmokers by P-adduct assay. (Meeting A b s t r a c t ) Proc. Am. Assoc. Cancer Res., 28, 400. Christman, J.K. (1982) Separation of major and minor deoxyribonucleoside monophosphates by reverse-phase high-performance l i q u i d chromatography. A simple method a p p l i c a b l e to q u a n t i t a t i o n of methylated n u c l e o t i d e s i n DNA. Ana l . Biochem., 119, 38-48. Day, R.S., Z i o l k o w s k i , C.H.J., S a i d i e r o , D.A., Meyer, S.A., and Mattern, M.R. (1980) Human tumor c e l l s t r a i n s d e f e c t i v e i n the r e p a i r of a l k y l a t i o n damage. Carcinogenesis, 1, 21-32. Demple, B., Jacobsson, A., Olsson, M., Robins, P., and L i n d a h l , T. (1982) Repair of a l k y l a t e d DNA i n Escherichia coli.: p h y s i c a l p r o p e r i t e s of 0^-methylguanine-DNA methyl-transferase. J . B i o l . Chem., 257, 13776-13780. Dolan, M.E., Morimoto, K., and Pegg, A.E. (1985) Reduction of 0 6 - a l k y l g u a n i n e -DNA a l k y l t r a n s f e r a s e a c t i v i t y i n HeLa c e l l s t r e a t e d w i t h 0 -alky1guanines. Cancer Res., 45, 6413-6417. D o l l , R., Payne, P., and Waterhouse, J . , eds. Cancer Incidence i n F i v e Continents. Geneva, S w i t z e r l a n d . IARC V o l . 1, 1966. D o l l , R., and Peto, R. (1981) The causes of Cancer: q u a n t i t a t i v e estimates of avoidable r i s k s of cancer i n the U n i t e d States today. J . N a t l . Cancer I n s t . , 66, 1192-1308. Domoradziki, J . , Pegg, A.E., Dolan, M.E., Maher, V.M., and McCormick, J . J . (1984) C o r r e l a t i o n between 0 -methylguanine DNA methyltransferase a c t i v i t y and r e s i s t a n c e of human c e l l s to the c y t o - t o x i c and mutagenic e f f e c t of N-methyl-N'-n i t r o - N - n i t r o s o g u a n i d i n e . Carcinogenesis, 5, 1641-1647. Doniger, J . , Day, R.S., DiPaolo, J.A. (1985) Q u a n t i t i a t i v e assessment of the r o l e of 0^-methylguanine i n the i n i t i a t i o n of carcinogenesis by methylating agents. Proc. N a t l . Acad. S c i . USA, 82, 421-425. Dunn, B.P., and S t i c h , H.F. (1986) 3 2 P - P o s t l a b e l i n g a n a l y s i s of aromatic DNA adducts i n human o r a l mucosal c e l l s . Carcinogenesis, 7, 1115-1120. 95 Dunn, B.P., Black, J . J . , and Maccubbin, A. (1987) J Z P - P o s t l a b e l i n g a n a l y s i s of aromatic DNA adducts i n f i s h from p o l l u t e d areas. Cancer Res., 47, 6543-6548. Dunn, B.P., and San, R.H.C. (1988) HPLC enrichment of hydrophobic DNA-carcinogen adducts f o r enhanced s e n s i t i v i t y of P - p o s t l a b e l i n g a n a l y s i s . Carcinogenesis, submitted f o r p u b l i c a t i o n . Everson, R.B., Randerath, E., S a n t e l l a , R.M., Ce f a l o , R.C., A v i t t s , T.A., and Randerath, K. (1986) D e t e c t i o n of smoking-related covalent DNA adducts i n human p l a c e n t a . Science, 231, 54-57. Everson, R.B., Randerath, E., A v i t t s , T.A., Schut, H.A.J., and Randerath, K. (1987) P r e l i m i n a r y i n v e s t i g a t i o n s of t i s s u e s p e c i f i c i t y , species s p e c i f i c i t y and s t r a t e g i e s f o r i d e n t i f y i n g chemicals causing DNA adducts i n human pl a c e n t a . Prog. exp. Tumor Res., 31, 86-103. Everson, R.B., Randerath, E., Haley, N.J., A v i t t s , T.A., and Randerath, K. (1987) Q u a n t i t i a t i v e a s s o c i a t i o n s between smoking and DNA adducts i n human p l a c e n t a measured by P - p o s t l a b e l i n g . (Meeting A b s t r a c t ) Proc. Am. Assoc. Cancer Res., 28, 1003. E t a i x , E., and Orgel, L.E. (1978) Phosphorylation of nucleosides i n aqueous s o l u t i o n u s i n g trimetaphosphate: formation of nucleoside t r i p h o s p h a t e s . J . Carbohydrates, Nucleosides, and Nu c l e o t i d e s , 5, 91-110. Farmer, P.B., Fos t e r , A.B., Jarman, M., a n d T i s d a l e , J . (1973) The a l k y l a t i o n of 2'-deoxyguanosine and of thymidine w i t h diazoalkanes-some observations on O - a l k y l a t i o n . Biochem. J . , 135, 203-213. F e n n e l l , T.R., J u h l , U., M i l l e r , E.C., and M i l l e r , J.A. (1986) I d e n t i f i c a t i o n and q u a n t i t a t i o n of h e p a t i c DNA adducts formed i n B6C3F^ mice from 1'-hydroxy-2',3'-dehydroestragole: comparison of the adducts detected w i t h 1'- H l a b e l l e d carcinogen and by P - p o s t l a b e l i n g . Carcinogenesis, 7, 1881-1887. F e n n e l l , T.R., Deal, F.H., and Swenberg, J.A. (1987) A n a l y s i s of formaldehyde-DNA adducts by P - p o s t l a b e l i n g (Meeting A b s t r a c t ) Proc. Am. Assoc. Cancer Res., 28, 370. Fowler, K.W., Buchi, G., and Essigmann, J.M. (1982) Synthesis and c h a r a c t e r i z a t i o n of an o l i g o n u c l e o t i d e c o n t a i n i n g a carcinogen-modified base, 0 6-methylguanine. J . Am. Chem. S o c , 104, 1050-1054. Gaffney, B.L., Marky, L.A., and Jones, R.A. (1984) Synthesis and c h a r a c t e r i z a t i o n of a set of four dodecadeoxyribonucleoside undecaphosphates c o n t a i n i n g O^-methylguanine opposite adenine, c y t o s i n e , guanine, and thymine. Biochemistry, 23, 5686-5691. Garner, R.C. (1985) Assessment of carcinogen exposure i n man. Carcinogenesis, 6, 1071-1078. Gerchman, L.L., and Ludlum, D.B. (1973) The polymerase p r o p e r t i e s of 0 -methylguanine i n templates f o r RNA. Biochim. Biophys. A c t a , 308, 310-316. Goth-Goldstein, R. (1980) I n a b i l i t y of Chinese hamster ovary c e l l s to exc i s e 0 6 - a l k y l g u a n i n e . Cancer Res., 40, 2623-2624. 96 Grafstrom, R.C., Pegg, A.E., Trump, B.F., and H a r r i s , C.C. (1984) Ch-a l k y ! guanine -DNA a l k y l t r a n s f e r s a s e a c t i v i t y i n normal human t i s s u e s and c e l l s . Cancer Res., 44, 2855-2857. Gupta, R.C., Reddy, M.V., and Randerath, K. (1982) 3 2 P - p o s t l a b e l i n g a n a l y s i s of n o n - r a d i o a c t i v e aromatic carcinogen-DNA adducts. Carcinogenesis, 3, 1081-1092. Gupta, R.C. (1984) Non-random b i n d i n g of the carcinogen N-hydroxy-2-acetyl-aminofluorene to r e p e t i t i v e sequences of r a t l i v e r DNA i n v i v o . Proc. N a t l . Acad. S c i . USA, 81, 6943-6947. 32 Gupta, R.C. (1985) Enhanced s e n s i t i v i t y of P - p o s t l a b e l i n g a n a l y s i s of aromatic carcinogen:DNA adducts. Cancer Res., 45, 5656-5662. Gupta, R.C, Dighe, N.R. , Randerath, K. , and Smith, H.C. (1985) D i s t r i b u t i o n of i n i t i a l and p e r s i s t e n t 2-acetylaminofluorene induced DNA adducts w i t h i n DNA loops. Proc. N a t l . Acad. S c i . USA, 82, 6605-6608. H a l l , J.A., and S a f f h i l l , R. (1983) The i n c o r p o r a t i o n of 0 6-methylguanosine and 04-methyldeoxythymidine monophosphates i n t o DNA by DNA polymerases I and a. N u c l e i c A c i d Res., 11, 4185-4193. H a l l , J . , B r e s i l , H., and Montesano, R. (1985) 0 6 - a l k y l g u a n i n e DNA a l k y 1 t r a n s f e r a s e a c t i v i t y i n monkey, human and r a t l i v e r . Carcinogenesis, 6, 209-211. H a r r i s , C.C. Role of carcinogens, cocarcinogens and host f a c t o r s i n human cancer r i s k . In: H a r r i s , C.C, and Autrup, H. , eds. Human Carcinogenesis. New York: Academic Press., 1983. pp 941-970. H a r r i s , C.C. (1985) Future d i r e c t i o n s i n the use of DNA adducts as i n t e r n a l dosimeters f o r monitoring human exposure to environmnental mutagens and carcinogens. Environ. Health. Perspect., 62, 185-191. H a r r i s , C.C, Vahakangas, K. , Newman, M.J. (1985) D e t e c t i o n of benzo(a)pyrene diol-epoxide-DNA adducts i n p e r i p h e r a l blood lymphocytes and a n t i b o d i e s to the adducts i n serum from coke oven workers. Proc. N a t l . Acad. S c i . USA, 82, 6672-6676. Heidelberger, C , and Davenport, G.R. (1961) L o c a l f u n c t i o n a l components of car c i n o g e n e s i s . Acta Un. I n t . Cancer, 17, 55-63. Hemminki, K. (1983) N u c l e i c a c i d adducts of chemical carcinogens and mutagens. Arch. T o x i c o l . , 52, 249-285. Herron, D.C, and Shank, R.C. (1979) Q u a n t i t a t i v e high-pressure l i q u i d chromatographic a n a l y s i s of methylated purines i n DNA of r a t s t r e a t e d w i t h chemical carcinogens. Anal. Biochem., 100, 58-63. Herron, D.C, and Shank, R.C. (1980) Methylated purines i n human l i v e r DNA a f t e r probable dimethylnitrosoamine poisoning. Cancer Res., 40, 3116-3117. 97 Higginson, J . , and Muir, C.S. (1979) Environmental c a r c i n o g e n e s i s : misconceptions and l i m i t a t i o n s to cancer c o n t r o l . J . N a t l . Cancer I n s t . , 63, 1291-1298. Hoffman, D., and Hecht, S.S. (1985) N i c o t i n e - d e r i v e d N-nitrosamines and t o b a c c o - r e l a t e d cancer: c u r r e n t s t a t u s and f u t u r e d i r e c t i o n s . Cancer Res., 45, 935-944. IARC Monographs on the E v a l u a t i o n of the Carcinogenic R i s k of Chemicals to Humans. Some N-Nitroso Compounds. V o l 17. (1978) pp 227-255. J e l i n e k , J . , K l e i b l , K., Dexter, T.M., and Margison, G.P. (1988) T r a n s f e c t i o n of murine m u l t i - p o t e n t haemopoietic stem c e l l s w i t h an E. coli DNA a l k y l t r a n s f e r a s e gene confers r e s i s t a n c e to the t o x i c e f f e c t s of a l k y l a t i n g agents. Carcinogenesis, 9, 81-87. K l e i h u e s , P., and Margison, G.P. (1974) C a r c i n o g e n i c i t y of N-methyl-N-n i t r o s o u r e a . P o s s i b l e r o l e of e x c i s i o n r e p a i r of 0 -methylguanine from DNA. J . N a t l . Cancer I n s t . , 53, 1839-1841. K l e i h u e s , P., and Bucheler, J . (1977) Long-term p e r s i s t e n c e of 0^-methylguanine i n r a t b r a i n DNA. Nature, 269, 625-626. Knudson, A.G., J r . Genetic p r e d i s p o s i t i o n to cancer. In: H i a l t , H.H., Watson, J.D., and Winston, J.A., eds. O r i g i n s of Human Cancer, Book A. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1977. pp 45-52. K o l o n e l , L.N., Hinds, M.W., and Hankin, J.H. Cancer p a t t e r n s among migrant and n a t i v e - b o r n Japanese i n Hawaii: r e l a t i o n to smoking, d r i n k i n g , and d i e t a r y h a b i t s . I n : Gelboin, H.V. et a l . , eds. Genetic and Environmental Factors i n Experimental and Human Cancers. Tokyo: Japan S c i e n t i f i c S o c i e t i e s Press, 1980. pp 327-340. K r i e k , E., Engelse, L.D., Scherer, E., and Westra, J.G. (1984) Formation of DNA m o d i f i c a t i o n s by chemical carcinogens. I d e n t i f i c a t i o n , l o c a l i z a t i o n and q u a n t i f i c a t i o n . Biochim. et Biophys. Acta, 738, 181-201. Kyrtopoulos, S.A., Vrotsou, B., Golematis, B., Bonatsos, M., and L a k i o t i s , G. (1984) 0 -methylguanine-DNA transmethylase a c t i v i t y i n e x t r a c t s of human g a s t r i c mucosa. Carcinogenesis, 5, 943-947. Loechler, E.L., Green, C.L., and Essignmann, J.M. (1984) In v i v o mutagenesis by 0 -methylguanine b u i l t i n t o a unique s i t e i n a v i r a l genome. Proc. N a t l . Acad. S c i . USA, 81, 6271-6275. Lohman, P.H.M., Jansen, J.D., and Baan, R.A. In: B e r l i n , A., Draper, M., Hemminki, K., and V a i n i o , H. eds. M o n i t o r i n g human exposure to carcinogens and mutagenic agents. Lyon, France. IARC S c i e n t i f i c P u b l i c a t i o n s No. 59, 1984, pp. 259-278. Loveless, A. (1969) P o s s i b l e relevance of 0-6 a l k y l a t i o n of deoxyguanosine to mutagenicity and c a r c i n o g e n i c i t y of nitrosamines and nitrosoamides. Nature, 223, 206-207. 98 Lu, L.-J.W., Disher, R.M., Reddy, M.V., and Randerath, K. (1986) J Z P -P o s t l a b e l l n g assay i n mice of t r a n s p l a c e n t a l DNA damage induced by the environmental carcinogens s a f r o l e , 4-aminobiphenyl, and benzo(a)pyrene. Lutz, W.K. (1979) I n v i v o covalent b i n d i n g of organic chemicals to DNA as a q u a n t i t a t i v e i n d i c a t o r i n the process of chemical c a r c i n o g e n e s i s . Mutat. Res., 65, 289-359. M a n i a t i s , T., F r i t s c h , E.F., and Sambrook, J . (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbour L a b o r a t o r i e s , New York 1982. Margison, G.P., and K l e i h u e s , P. (1975) Chemical car c i n o g e n e s i s i n the nervous system. P r e f e r e n t i a l accumulation of 0 -methylguanine i n r a t b r a i n d e o x y r i b o n u c l e i c a c i d during r e p e t i t i v e a d m i n i s t r a t i o n of N-methyl-N-n i t r o s o u r e a . Biochem. J . , 148, 521-525. Maugh, T.H. (1984) Tracking exposure to t o x i c substances. Science, 226, 1183-1184. M i l l e r , E.C., and M i l l e r , J.A. (1981) Searches f o r u l t i m a t e carcinogens and t h e i r r e a c t i o n s w i t h c e l l u l a r macromolecules. Cancer, 47, 2327-2345. M i t r a , S., Palm, B.C., and Foote, R.S. (1982) 0 6-methylguanine-DNA methy l t r a n s f e r a s e i n w i l d - t y p e and ada mutants of Escherichia coli. J . B a c t e r i d . , 152, 534-537. Moloney, S.J., W i e l b k i n , P., Cummings, S.W., and Prough, R.A. (1985) Metabolic a c t i v a t i o n of the t e r m i n a l N-methyl group of N-isopropyl-alpha,(2-methyl-hydrazine)-p-toluamide h y d r o c h l o r i d e (procarbazine). Carcinogenesis, 6, 397-401. Montesano, R., B r e s i l , H., and Margison, G.P. (1979) Increased e x c i s i o n of 0^-methylguanine from r a t l i v e r DNA a f t e r c hronic a d m i n i s t r a t i o n of dimethylnitrosamine. Cancer Res., 39, 1789-1802. Morimoto, K., Dolan, M.E., S c i c c h i t a n o , D., and Pegg, A.E. (1985) Repair of 0 -propylguanine and 0 -butylguanine i n DNA by 0 -alkylguanine-DNA a l k y l t r a n s f e r a s e from r a t l i v e r and E. coli. Carcinogenesis, 6, 1027-1031. M o r r i s , S.M., Beranek, D.T., and H e f l i c h , R.H. (1983) The r e l a t i o n s h i p between s i s t e r - c h r o m a t i d exchange i n d u c t i o n and the formation of s p e c i f i c methylated DNA adducts i n Chinese hamster ovary c e l l s . Mutat. Res., 121, 261-266. Newbold, R.F., Warren, W., Medcalf, A.S.C., and Amos, J . (1980) Mutagenecity of c a r c i n o g e n i c methylating agents i s a s s o c i a t e d w i t h a s p e c i f i c DNA m o d i f i c a t i o n . Nature, 283, 596-599. Olsson, M., and L i n d a h l , T. (1980) Repair of a l k y l a t e d DNA i n E s c h e r i c h i a c o l i : methyl group t r a n s f e r from 06-methylguanine to a p r o t e i n c y s t e i n e r e s i d u e . J . B i o l . Chem., 255, 10569-10571. Parsa, I . , Friedman, S., and Cleary, CM. (1987) V i s u a l i z a t i o n of 0 6-methylguanine i n t a r g e t c e l l n u c l e i of di m e t h y l n i t r o s a m i n e - t r e a t e d human pancreas by a murine monoclonal antibody. Carcinogenesis, 8, 839-846. 99 Parthasarathy, R., and F r i d e y , S.M. (1986) Conformation of 0 -alkylguanosines: molecular mechanism of mutagenesis. Carcinogenesis, 7, 221-227. Pegg, A.E. (1984) M e t h y l a t i o n of the 0^ p o s i t i o n of guanine i n DNA i s the most l i k e l y i n i t i a t i n g event i n carcinogenesis by methylating agents. Cancer Invest., 2, 223-231. Perera, F.P. (1987) Molecular cancer epidemiology: A new t o o l i n cancer prevention. J . N a t l . Cancer I n s t . , 78, 887-898. P h i l i p p , M., and S e l i g e r , H. (1977) Spontaneous ph o s p h o r y l a t i o n of nucleosides i n formamide-ammonium phosphate mixtures. Naturwissenschaften, 64, 273. P h i l l i p s , D.H., Hemminki, K., Hewer, A., and Grover, P.L. (1987) 3 2 P -P o s t l a b e l i n g of white b l o o d c e l l DNA from foundry workers. (Meeting A b s t r a c t ) Proc. Am. Assoc. Cancer Res., 28, 403. Ramos, D.L., and S c h o f f s t a l l , A.M. (1983) Reversed-phase high-performance l i q u i d chromatographic s e p a r a t i o n of nucleosides and n u c l e o t i d e s . J . Chromatography, 261, 83-93. Randerath, E., Yu, C-T., and Randerath, K. (1972) Base a n a l y s i s of r i b o p o l y n u c l e o t i d e s by chemical t r i t i u m l a b e l i n g . A methodological study w i t h model n u c l e o s i d e s , and p u r i f i e d tRNA species. Anal. Biochem., 48, 172-198. Randerath, E., Agrawal, H.P., Weaver, J.A., Bordelon, C.B., and Randerath, K. (1985) P - P o s t l a b e l i n g a n a l y s i s of DNA adducts p e r s i s t i n g f o r up to 42 weeks i n the s k i n , epidermis, and dermis of mice t r e a t e d t o p i c a l l y w i t h 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 6, 1117-1126. Randerath, E., A v i t t s , T.A., Reddy M.V., M i l l e r , R.H., Everson, R.B., and Randerath, K. (1986) Comparative P-Analysis of c i g a r e t t e smoke-induced DNA damage i n human t i s s u e s and mouse s k i n . Cancer Res., 46, 5869-5877. Randerath, K., and Randerath, E. (1964) Ion-exchange chromatography of n u c l e o t i d e s on p o l y ( e t h y l e n e ) - i m i n e - c e l l u l o s e t h i n l a y e r s . J . Chromatography, 16, 111-125. Randerath, K., Reddy, M.V., and Gupta, R.C. (1981) 3 2 P - L a b e l i n g t e s t f o r DNA damage. Proc. N a t l . Acad. S c i . USA, 78, 6126-6129. Randerath, K., Reddy, M.V., and Disher, R.M. (1986) Age- and t i s s u e - r e l a t e d DNA m o d i f i c a t i o n s i n untreated r a t s : d e t e c t i o n by P - p o s t l a b e l i n g assay and p o s s i b l e s i g n i f i c a n c e f o r spontaneous tumor i n d u c t i o n and aging. Carcinogenesis, 7, 1615-1617. Randerath, K., A v i t t s , T.A., M i l l e r , R.H., and Randerath, E. (1987) 3 2 P -P o s t l a b e l i n g a n a l y s i s of c i g a r e t t e smoking-related DNA adducts i n t a r g e t t i s s u e s of human ca r c i n o g e n e s i s . (Meeting A b s t r a c t ) Proc. Am. Assoc. Cancer Res., 28, 388. Reddy, M.V. , Gupta, R.C, and Randerath, K. (1981) 3 2P-base a n a l y s i s of DNA. Anal. Biochem., 117, 271-279. 100 Reddy, M.V., Gupta, R.C., Randerath, E., and Randerath, K. (1984) °^P-P o s t l a b e l i n g t e s t f o r covalent DNA b i n d i n g of chemicals i n v i v o : a p p l i c a t i o n to a v a r i e t y of aromatic carcinogens and methylating agents. Carcinogenesis, 5, 231-243. Reddy, M.V., and Randerath, K. (1986) Nuclease Pl-mediated enhancement of s e n s i t i v i t y of P - p o s t l a b e l i n g t e s t f o r s t r u c t u r a l l y d i v e r s e DNA adducts. Carcinogenesis, 7, 1543-1551. Renard, A.. and V e r l y , W. (1980) A chromatin f a c t o r i n r a t l i v e r which destroys O - e t h y l g u a n i n e i n DNA. FEBS l e t t . , 114, 98-102. R e z n i k o f f , C.A., Bertram, J.S., Brankow, D.W., and Heidelberger, C. (1973) Q u a n t i t a t i v e and q u a l i t a t i v e s t u d i e s of chemical t r a n s f o r m a t i o n of cloned C3H mouse-embryo c e l l s s e n s i t i v e to post-confluence i n h i b i t i o n of c e l l d i v i s i o n . Cancer Res., 33, 3239-3249. Richardson, K.K., Richardson, F.C., Crosby, R.M., Swenberg, J.A., and Skopek, T.R. (1987) DNA base changes and a l k y l a t i o n f o l l o w i n g i n v i v o exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea. Proc. N a t l . Acad. S c i . USA, 84, 344-348. Rydberg, B. and L i n d a h l , T. (1982) Non-enzymatic m e t h y l a t i o n of DNA by the i n t r a c e l l u l a r methyl group donor S-adenosyl-L-methionine i s a p o t e n t i a l l y mutagenic r e a c t i o n . EMBO J . , 1, 211-216. S a f f h i l l , R., and H a l l , J . (1981) A convenient p r e p a r a t i o n of i s o m e r i c a l l y pure nucleoside 5'-monophosphates from unprotected n u c l e o s i d e s . J . Carbohydrates, Nucleosides and N u c l e o t i d e s , 8, 573-583. Samson, L., and C a i r n s , J . (1977) A new pathway f o r DNA r e p a i r i n Escherichia coli. Nature, 267, 281-282. Samson, L., D e r f l e r , B., and Waldstein, E.A. (1986) Suppression of human DNA a l k y l a t i o n - r e p a i r defects by Escherichia coli DNA-repair genes. Proc. N a t l . Acad. S c i . USA, 83, 5607-5610. Sasaki, T., and Yoshida, T. (1935) Experimentelle erzeugang des lebercarcinoma durch f u t t e r u n g mit 0-amidoazotoluol. Virchows Arch. f. p a t h o l . Anat., 295, 175-200. S c h o f f s t a l l , A.M., (1976) P r e b i o t i c p hosphorylation of nucleosides i n formamide. O r i g i n s of L i f e , 7, 399-412. S c h o f f s t a l l , A.M., and Kokko, B., (1978) Nucleoside p h o s p h o r y l a t i o n i n amide s o l u t i o n s . O r i g i n s of L i f e , 193-198. S c h o f f s t a l l , A.M., Barto, R.J., and Ramos, D.L., (1982) Nucleoside and deoxynucleoside phosphorylation i n formamide s o l u t i o n s . O r i g i n s of L i f e , 12, 142-151. S c h o f f s t a l l , A.M., and Laing, E.M., (1984) E q u i l i b r a t i o n of n u c l e o t i d e d e r i v a t i v e s i n formamide. O r i g i n s of L i f e , 14, 221-228. 101 S c i c c h i t a n o , D., and Pegg, A.E. (1982) K i n e t i c s of r e p a i r of 0 -methylguanine i n DNA by 0 -methylguanine DNA methyltransferase i n v i t r o and i n v i v o . Biochem. Biophys. Res. Commun., 109, 995-1001. Se a r l e , C.E. ed. Chemical Carcinogens (2 Ed.) ACS Monograph 182 Washington, D.C.: ACS. 1984. S h i l o h , Y., and Becker, Y. (1981) K i n e t i c s of 0^-methylguanine r e p a i r i n human normal and a t a x i a t e l a n g i e c t a s i a c e l l l i n e s and c o r r e l a t i o n of r e p a i r c a p a c i t y w i t h c e l l u l a r s e n s i t i v i t y of methylating agents. Cancer Res., 41, 5114-5120. Singer, B. (1985) I n v i v o formation and p e r s i s t e n c e of m o d i f i e d nucleosides r e s u l t i n g from a l k y l a t i n g agents. Environ. Health Perspect., 62, 41-48. Singer, G.M., Chuan, J . , Roman, J . , Min-Hsin, L., and L i j i n s k y , W. (1986) Nitrosamines and nitrosamine precursors i n foods from L i n x i a n , China, a h i g h incidence area f o r esophageal cancer. Carcinogenesis, 7, 733-736. Sober, H.A., ed., Handbook of Biochemistry. S e l e c t e d Data f o r Molecular B i o l o g y . Cleveland, Ohio: Chemical Rubber Co. pp H45-H46, S t r i c k l a n d , P.T., and Boyle, J.M. Immunoassay of carcinogen m o d i f i e d DNA. In: Cohn, ed. Progress i n n u c l e i c a c i d research and molecular b i o l o g y , v o l 31. New York: Academic Press. 1984 pp 1-58. Sueoka, N., and Cherry, T. (1967) F r a c t i o n a t i o n of DNA on methylated albumin column. Methods Enzymol., 12, 562-566. Sulkowski, E., Bjork, W., and Laskowski, M., (1963) A s p e c i f i c and n o n s p e c i f i c a l k a l i n e monophosphatase i n the venom of Bothrops a t r o x and t h e i r occurrence i n the p u r i f i e d venom phosphodiesterase. J . B i o l . Chem, 238, 2477-2486. Swenberg, J.A., and B e d e l l , M.A. C e l l - s p e c i f i c DNA a l k y l a t i o n and r e p a i r : a p p l i c a t i o n of new f l u o r i m e t r i c techniques to detect adducts. In: Bridges, Butterworth.and Weinstein, eds. I n d i c a t o r s of genotoxic exposure. New York: Cold Spring Harbor L a b o r a t o r i e s , 1982. pp 205-220. Toorchen, D., and Topal, M.G. (1983) Mechanism of chemical mutagenesis and c a r c i n o g e n e s i s : e f f e c t s on DNA r e p l i c a t i o n of m e t h y l a t i o n at the 0^-guanine p o s i t i o n of dGTP. Carcinogenesis, 4, 1591-1597. Topal, M.D., and Baker, M.S. (1982) DNA precursor p o o l : A s i g n i f i c a n t t a r g e t f o r N-methyl-N-nitrosourea i n C3H10T1/2 clone 8 c e l l s . Proc. N a t l . Acad. S c i . USA, 79, 221-2215. Umbenhauer, D. , Wild, C P . , Montesano, R. , S a f f h i l l , R. , Boyle, J.M., Huh, N. , K i r s t e i n , U., Thomale, J l , Rajewsky, M.F., and Lu, S.H. (1985) 0 -methyldeoxyguanosine i n oesophageal DNA among i n d i v i d u a l s at h i g h r i s k of oesophageal cancer. I n t . J . Cancer, 36, 661-665. Vahakangas, K., T r i v e r s , G., Rowe, M., and H a r r i s , C.C. (1985) Benzo(a)pyrene diolepoxide-DNA adducts detected by synchronous fluorescence spectrophotometry. Environ. Health Perspect, 62, 101-104. 102 Waterhouse, J . , Muir, C , Shanmugaratnam, K., and Powell, J . , eds. Cancer Incidence i n Fi v e Continents, V o l 4. IARC S c i e n t i f i c P u b l i c a t i o n s No. 42, Lyon, France: IARC, 1982. Watson, W.P. (1987) P o s t - r a d i o l a b e l l i n g f o r d e t e c t i n g DNA damage. Mutagenesis, 2, 319-331. Watson, W.P., Crane, A.E Davies, R., Smith, R.J., and Wright, A.S. (1987) A p o s t l a b e l i n g assay f o r N -(2-oxoethy1)guanine, the p r i n c i p a l v i n y l c h l o r i d e -DNA adduct. A r c h i v . T o x i c o l . , Suppl. 11, 89-92. W i l d C P . , Smart, G. , S a f f h i l l , R. , and Boyle, J.M. (1983) Radioimmunoassay of 0 -methyldeoxyguanosine i n DNA of c e l l s a l k y l a t e d i n v i t r o and i n v i v o . Carcinogenesis, 4, 1605-1609. Wi l d , C P . , Umbenhauer, D., Chapot, B., and Montesano, R. (1986) Monitoring of i n d i v i d u a l human exposure to a f l a t o x i n (AF) and N-nitrosamines (NNO) by immunoassays. J . C e l l . Biochem., 30, 171-179. W i l l i a m s , L.D., and Shaw, B.R. (1987) Protonated base p a i r s e x p l a i n the ambiguous p a i r i n g p r o p e r t i e s of 0 -methylguanine. Proc. N a t l . Acad. S c i . USA., 84, 1779-1783. World Health O r g a n i z a t i o n . Tobacco Habits other than Smoking: B e t e l - q u i d and areca-nut chewing, and some r e l a t e d nitrosamines. IARC monographs on the e v a l u a t i o n of the ca r c i n o g e n i c r i s k of chemicals to humans. 1985 Wilson, V.L., Smith, R.A., Autrup, H., Krokan, H., Musci, D.E., Le, N-N-T., Longoria, J . , Z i s k a , D., and H a r r i s , C.C. (1986) Genomic 5-methylcytosine determination by P - p o s t l a b e l i n g a n a l y s i s . Anal. Biochem., 152, 275-284. Wogan, G.N., and G o r e l i c k , N.J. (1985) Chemical and biochemical dosimetry of exposure to genotoxic chemicals. Environ. Health Perspect., 62, 5-18. Wong, D., M i t c h e l l , C.E., Wolff, R.K., Mauderly, J.L., and J e f f r e y , A.M. (1986) I d e n t i f i c a t i o n of DNA damage as a r e s u l t of exposure of r a t s to d i e s e l engine exhaust. Carcinogenesis 7, 1595-1597. Yamagiwa, K., and Ichakawa, K. (1915) Experimentelle s t u d i e uber d i e pathologenese der e p i t h e l i a l g e s c h wulste. M i t t e r l u n g e n Med. F a c u l t a t . K a i s e r l . Univ., 15, 295-344. Yarosh, D.B. (1985) The r o l e of O^-methylguanine-DNA methyltransferase i n c e l l s u r v i v a l , mutagenesis and carc i n o g e n e s i s . Mut. Res., 145, 1-16. Z a r b l , H., Sukumar, S., Arth u r , A.V., Martin-Zanca, D., and Barbaeid, M. (1985) D i r e c t mutagenesis of Ha-ras-1 oncogenes by N-nitroso-methylurea during i n i t i t i a t i o n of mammary carcinogenesis i n r a t s . Nature, 315, 382-385. 

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