.- .. I E.- .- III-Elli THESIS Date 0-7639 LIBRARY Michigan Stat: University ——--.-— -— --—'-—' This is to certify that the thesis entitled QUALITATIVE AND QUANTITATIVE ASSESSMENTS OF THE ALKYLATION OF DNA IN SPECIFIC HEPATIC CHROMATIN FRACTIONS FOLLOWING EXPOSURE TO METHYLNITROSOUREA OR DIMETHYLNITROSAMINE presented by Elaine Marie Faustman has been accepted towards fulfillment of the requirements for Ph.D. Pharmacology and de e in gre Toxicology I /7 .-) . .1" //l C/ £6 ’ A; / / Major professor Jan. 7, 1981 . p1,, flank; k' ‘. {tum '_ j" (Timmy. ' ‘I' ‘ ,l‘.‘_. . Zlv" OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remv charge from circulation recor QUALITATIVE AND QUANTITATIVE ASSESSMENTS OF THE ALKYLATION OF DNA IN SPECIFIC HEPATIC CHROMATIN FRACTIONS FOLLOWING EXPOSURE TO METHYLNITROSOUREA OR DIMETHYLNITROSAMINE By Elaine Marie Faustman A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1980 ABSTRACT Qualitative and Quantitative Assessments of the Alkylation of DNA in Specific Hepatic Chromatin Fractions Following Exposure to Methylnitrosourea or Dimethylnitrosamine by Elaine Marie Faustman The investigations reported in this thesis have assessed qualita- tively and quantitatively the DNA alkylation induced by dimethylni- trosamine (DMN) and methylnitrosourea (MNU), two carcinogenic methyl- ating agents. Male Sprague-Dawley rats were exposed to (BR—methyl)MNU, 15 umoles, 10 uCi/umole/lOO g or (lac-methy1)DMN, 8 umoles, 1.0 uCi/ umole/lOO g via gastric intubation. Alkylation of hepatic DNA was assessed at 3, 24, 72 and 168 hr post-intubation. Hepatic chromatin was fractionated into portions having characteristics of template active euchromatin and template repressed heterochromatin by digestion with DNase II followed by MgCl precipitation. 2 These studies indicate that initial DMN and MNU induced alkyla- tion of hepatic DNA occurs in a non-random fashion. The putative euchromatin fractions appear to be selectively alkylated. Since the rates of loss of alkylation from the heterochromatin fractions were less than euchromatin, differences between euchromatin and hetero- chromatin were probably a result of actual increases in initial alkylation of euchromatin DNA rather than faster repair capacities of heterochromatin DNA. Elaine Marie Faustman The rates of hepatic DNA synthesis observed following carcinogen exposure did not differ from rates of DNA synthesis seen in animals intubated with only the solvent. Thus, in these studies loss of alkyl groups from various chromatin regions was not due to carcinogen- induced necrosis and subsequent dilution of label by compensatory hyperplasia. The euchromatin/heterochromatin DNA alkylation ratio of MNU exposed rats is 2 times the alkylation ratio observed for DMN treated animals at times of peak alkylation. These observations suggest differences in the alkylation pattern produced by these two carcino- gens. These might be due to differences in the site of generation of the methylating species in the cellular environment, stability of the parent compound, as well as stability of the methylcarbonium ions produced. A high pressure liquid chromatography separation has been de— veloped which can separate 7-methylguanine, 06—methylguanine, 1— methyladenine, 3-methyladenine, guanine and adenine. This system which separates four of the major methylated DNA-purines observed after DMN or MNU exposure offers several advantages over systems currently in use: separation of bases in a simultaneous column run, requires only one column for separation and uses an isocratic rather than gradient elution system. Using this HPLC system the patterns of specific purine alkylation in total chromatin has been compared to the pattern of specific purine alkylation in heterochromatin at 24 hr following exposure to DMN or MNU. A qualitatively similar pattern of 7-methy1guanine, 06-methyl- guanine, 1-methyladenine and 3-methyladenine alkylation was observed Elaine Marie Faustman in total chromatin versus heterochromatin following carcinogen expo— sure. Possible differences in the pattern of alkylation of various genome regions can not be ruled out since subtle differences in the patterns of purine alkylation would not have been detectable in this system. Our finding that, initially, alkylation of euchromatin DNA occurs to a greater extent than alkylation of heterochromatin DNA is compa- tible with the observation of a qualitatively similar pattern of specific DNA-purine alkylation in total chromatin as compared to he— terochromatin regions. If the same reactive species is generated in both regions, one would expect a similar pattern of DNA-purine alkyl- ation. Selective alkylation of template—active chromatin regions could increase the probability of an alteration of phenotype as a result of aberrant RNA formation during transcription. Damage in heterochroma- tin DNA would not be expressed. The formation of a mutation in eu- chromatin, either through base mispairing during replication, deletion of a section of DNA to be replicated or as a result of an error—prone repair process, could also result in an alteration of phenotype. The derepression of previously quiescent regions of the genome through alterations in gene expression (possibly during the promotion stage of carcinogenesis) could result in initial carcinogen-induced modifi— cations of heterochromatin being manifest. ACKNOWLEDGEMENTS This thesis is dedicated to my husband, Neil R. Watts; to my mother, Alice M. Faustman; and to my great aunt, Cora C. Maupin. Their combined encouragement and support have made it possible for me to complete this work. I am indebted to Sandra Moore, Cathy Hironaka and Kimberly Thorton for their excellent technical assistance. I am also indebted to Diane Hummel for her assistance in the preparation of this manuscript. Dr. Jay Goodman has provided intellectual stimula— tion and guidance throughout my four years of study at Michigan State University and for this I sincerely thank him. ii ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTI N MATERIALS \DCDNwUIbWNH TABLE OF CONTENTS Background --— Carcinogenicity of Methylnitrosourea and Dimethylni- trosamine Chemistry of Carcinogen-induced Alkylation of DNA ------ Intragenomic Distribution of Carcinogen-induced Damage- Chromatin Fractionation Experimental Objectives AND METHODS Materials Animals, Carcinogen Administration Isolation of Chromatin Chromatin Fractionation Analysis of Carcinogen Binding to Macromolecules-—----- Assessment of DNA Synthesis Purification of DNA for Purine Isolation Isolation of Purines High Pressure Liquid Chromatographic Separation -------- RESULTS 1. \lC‘LflJ-‘DJN Alkylation Following Methylnitrosourea Exposure -------- Alkylation Following Dimethylnitrosamine Exposure ------ DNA Synthesis Assessment Characterization of DNA and Purine Fractions Purine Separation Alkylation of Purines in Hepatic DNA Purine Alkylation in Specific Chromatin Fractions ------ iii Page ii vi viii 13 22 26 27 27 27 28 29 30 31 31 32 33 35 35 4O 51 54 58 58 62 TABLE OF CONTENTS (continued) Page DISCUSSION 90 CONCLUSIONS 112 BIBLIOGRAPHY 117 iv Table 10 11 12 13 LIST OF TABLES Page kylation of Chromatin Nucleic Acids Following ( Hdmethyl)Methylnitrosourea Exposure 36 Characterization of Hepatic Chromatin 39 Alkylation of Chromatin Nucleic Acids Following (14C— methyl)Dimethylnitrosamine Exposure 43 Comparison of DNA Yield Following Various Isolation Procedures 55 Recovery of Purines from DNA Isolated Using a Modified Marmur Procedure 56 Characterization of Purine Hydrolysate Used for HPLC--- 57 Methylated Purines in DNA Isolated from Hepatic Chroma- tin of Rats Treated with H—MNU 61 Analysis of Purine Methylation in DNA of Hepatic Chro- matin Isolated from Rats Treated with H-MNU 63 Rate of Loss of Methylated Bases from Hepatic Chromatin 74 Purine Methylation in DNA from Total Chrzmatin and Heterochromatin Following Exposure to Cl -DMN 78 Purine Methylation in DNA from Total Chromatin and Heterochromatin Following Exposure to H-MNU 81 Comparison of 7-MeG Half Lives Determined by Various 96 Investigative Groups Comparison of 06-MeG Half Lives Determined by Various Investigative Groups 97 Figure 10 11 12 13 14 15 16 LIST OF FIGURES Page Structure of Dimethylnitrosamine and Methylnitrosourea- 6 Proposed Generation of Methylating Intermediates from Dimethylnitrosamine and N-methyl-N-nitrosourea --------- 7 Major Ig_Vivo Sites of Carcinogen—induced Purine Methylation 10 Diagramatic Representation of Chromatin Fractionation by DNase II-Mgc12 Method 23 Time Course of Methylnitrosourea-induced Alkylation of Hepatic DNA 37 Methylnitrosourea-induced Alkylation of Specific Chroma- tin Fractions 41 Time Course of Dimethylnitrosamine-induced Alkylation of Hepatic DNA 45 Dimethylnitrosamine-induced Alkylation of Specific Chromatin Fractions 47 Comparison of MNU— and DMN-induced Alkylation of DNA--- 49 Assessment of the Degree of Carcinogen-induced Hyper- plasia 52 Separation of Purines by High Pressure Liquid Chroma- tography --- 59 Alkylation of Purine Versus Apurinic Fractions --------- 64 Loss of 74Methy1guanine from Rat Hepatic DNA 66 Loss of 06-Methylguanine from Rat Hepatic DNA ---------- 68 Loss of 14Methyladenine from Rat Hepatic DNA 70 Loss of 3-Methy1adenine from Rat Hepatic DNA 72 vi LIST OF FIGURES (continued) Figure 17 18 19 20 21 22 Alkylation of Specific Methylated Bases in Total Chromatin Versus Heterochromatin DNA Following Expo- sure to Cl4—DMN Alkylation of Specific Methylated Basis in Total Chro— matin Versus Heterochromatin DNA Following Exposure to H-MNU Comparison of MNU- and DMN-induced Patterns of Purine Alkylation Identification of Methylated Bases in Chromatin Frac- tions at 3 hr Post-intubation of 3H-MNU r Analysis of Methylated Bases in Chromatin at 3 hr After 3H—MNU Exposure Biological Consequences of Carcinogen-induced DNA Damage in Euchromatin Versus Heterochromatin vii Page 76 79 82 85 87 110 hr DMN 7—MeG 06—MeG lAMeA 3-MeA P2 82 HPLC LIST OF ABBREVIATIONS hour methylnitrosourea dimethylnitrosamine Guanine 7—methy1guanine 06-methylguanine l-methyladenine 3-methyladenine heterochromatin euchromatin high pressure liquid chromatography adenine viii INTRODUCTION I. Background In 1914 T. Boveri proposed that an alteration of the chromatin complex of somatic cells was the critical event in carcinogenesis. Although this hypothesis was proposed prior to any knowledge of the structure of hereditary macromolecules within the chromatin complex, this idea has helped to form the foundation of the somatic mutation theory of carcinogenesis. Bauer in 1928 modified Boveri’s hypothesis to include mutation of genes as the possible alteration of chromatin that would lead to tumorigenesis. Many researchers now believe that the covalent binding of carcinogens to DNA within chromatin is the critical alteration that leads to mutations and some such muta- tion(s) has a causative role in neoplastic transformation. Most, if not all, chemical carcinogens yield electrophiles, either via metabolic activation or spontaneously, which bind covalent- ly to nucleophilic moieties in DNA, RNA and protein (Miller and Miller, 1947; Wheeler and Skipper, 1957; Marroquin and Father, 1962). Miller (1978) has recently reviewed evidence that the covalent binding of carcinogens to DNA may be the critical event in carcinogenesis. Interest in DNA developed since changes in DNA could explain the heritable and primarily irreversible characteristics of neoplastic growth. Brookes and Lawley (1964a,b) were able to demonstrate a strong positive correlation between the binding of radioactively 2 labelled polycyclic aromatic hydrocarbons to the DNA of mouse skin and the carcinogenic potency of the hydrocarbons as defined by Iball (1939). When Iball's index was used to rank carcinogens on the number of tumors produced and on the latency period until tumors developed, no correlation of carcinogen potency and binding to RNA or protein was observed, the correlation was specific for DNA. Burdette in 1955 reviewed earlier research investigating a possible correlation between the carcinogenic and mutagenic activities of chemicals. Auerbach (1939-40), Demerec 35.31. (1949) and Burdette (1955) failed to establish such a relationship. Later studies which accounted for the necessity of certain chemicals to be metabolized into an active form prior to exerting carcinogenic or mutagenic pro- perties resulted in establishing such a correlation (Maher §£_al., 1968; Miller and Miller, 1971; Ames 35 31., 1973). When carcinogens were tested in the Ames salmonella/microsomal test system, 90% of the carcinogens were mutagens (McCann g£_gl,, 1975). Other evidence suggesting that DNA is a critical target in the process of carcinogenesis is the increased incidence of skin cancer in xeroderma pigmentosa patients deficient in DNA excision repair capa- bilities (Cleaver, 1969). Correlations between the extent of repair and clinical manifestations of neoplasms could be made (Takebe st 21,, 1977). As well, if cancer occurs via a mutational mechanism, then cancer prone individuals might also be expected to exhibit increased susceptibility to mutations at all loci. Fibroblasts from xeroderma pigmentosa patients exhibited increased cytotoxicity and mutagenicity 3 upon U.V. or polycyclic hydrocarbon exposure in contrast to normal fibroblasts (Maher and McCormick, 1976; Maher gt al., 1977). In summary, evidence does exist indicating that interaction of carcinogens with DNA may represent one of the critical events in the transformation of a normal cell to a malignant state. Speculation on how covalent binding of carcinogens to DNA leads to the initial phase of carcinogenesis is still controversial, however, several theories have been proposed. Berenblum in 1941 noted that subcarcinogenic exposure to benzpyrene and subsequent croton oil exposures could increase the number of tumors produced in skin above levels seen with carcinogen or croton oil exposure alone. As well, decreases in the latency period for tumor development were produced when the subsequent croton oil exposures occurred. These phenomena did not occur when croton oil was given prior to carcinogen exposure. Further studies (Berenblum and Shubick, 1947a,b, 1949) demonstrated that only a single carcinogen exposure was necessary and that this exposure could result in "semi—permanent" alterations of a cell that could be induced 43 weeks later when croton oil applications were started. These data substantiated observations that carcinogenesis could be viewed as a progression of initiated cells by promoting influences into neoplastic states (Rous and Kidd, 1941; Friedwald and Rous, 1944). The two—stage theory of carcinogenesis states that first initiation, and then subsequently promotion leads to carcinogenesis (Berenblum, 1941; Friedwald and Rous, 1944; Berenblum and Shubick, 1947a,b, 1949). Initiation is pictured as the formation of heritable but unexpressed changes in the cell genome. Subsequently, promotion 4 causes the phenotypic expression of these changes in genotype as altered metabolism and later as altered cellular morphology and thus ultimately as a malignant tumor (Boutwell, 1974). Weinstein gt_al, (1979) have proposed several molecular mecha- nisms to explain how the process of initiation could occur following covalent binding of carcinogens to DNA. These include point muta- tions, abberations at the DNA level in genetic mechanisms that may normally control differentiation and abberations in epigenetic mecha— nisms or differentiation such as altered chromatin structure and DNA methylation (S—methylcytosine in DNA as a modifier of protein-DNA interaction and gene activity; Razin and Riggs, 1980). Boutwell (1974) has expanded on the proposal of Pitot and Heidel- berger (1963), that carcinogens could act by binding repressor genes thus inactivating the repressor effects and altering gene regulation. Boutwell (1974) states that promoters may be gene activators and thus regulate gene transcription. If promoter exposure does result in expression of previously repressed areas of the genome then it would be logical to expect to see enzyme induction (Boutwell, 1974). In— creases in the levels of ornithine decarboxylase (O'Brien and Diamond, 1977) and the protease plasminogen activation (Wigler and Weinstein, 1976; Weinstein, 1979) have been correlated to promoter activity. This section has provided background information to substantiate the importance of research involving the assessment of DNA alteration following carcinogen exposure. 5 II. Carcinogenicity of Methylnitrosourea and Dimethylnitrosamine Two carcinogens which have been investigated in the following studies are dimethylnitrosamine (DMN) and methylnitrosourea (MNU) (Figure 1). DMN has been shown to produce hepatocellular carcinomas in rats by dietary feeding or by a single administration (Magee and Barnes, 1956; Craddock, 1971; Craddock, 1975). MNU can initiate hepatocarcinogenesis (Craddock and Frei, 1974; Cayama g£_§13, 1978; Solt 35 31,, 1980) as well as produce hemangiosarcomas upon intrapor- tal infusion (Lijinsky £5 31,, 1972; Craddock and Frei, 1974). DMN requires metabolic activation whereas MNU spontaneously decomposes to form the reactive methylating species (Magee and Vandekar, 1958; Lawley, 1972, 1974) (Figure 2). III. Chemistry of Carcinogen-induced Alkylation of DNA MNU has been classified as an SNI type alkylating agent (Lawley, 1974). Reagents of this type typically have a low Swain-Scott S factor and thus react more extensively at oxygen atoms in DNA or RNA than SN2 type alkylating agents, methylmethanesulfonate or dimethyl sulphate. (The Swain-Scott S factor enables comparisons of the chemi- cal reactivity of various nucleophilic reagents to be made to the reactivity of the hydroxide ion [Swain and Scott, 1953]). DMN appears to methylate O-atom sites in DNA to a relatively greater extent than SN2 type carcinogens and thus also can be classified as a SNl like alkylating agent (Pegg and Nicoll, 1976). SNl type agents follow first-order kinetics and the rate of methylation is dependent not on the nucleophilic concentration but on the concentration of the alkyla- ting agent. This is in contrast to SN2 type agents where the N—N=O METHYLNITROSOUREA ’ CH,\ . ; N—N=O 'DIMETHYLNITROSAMINE, " CH3/ . Figure 1. Structure of dimethylnitrosamine and methylnitrosourea. Figure 2. Proposed pathways for generation of methylating interme- diates from dimethylnitrosamine and N-methyl-N-nitrosourea. (CH3)2N-N0 CH3N-N0CONH2 ENZYMIC + NONENZYMIC NADPHl 02 + 0H- CH3 NHNO l CH3N2+ OH' J. + CH3 1. CH3R Figure 2 9 methylation reaction is dependent on the nucleophilicity of the site and on the concentration of the alkylating agent (Lawley, 1974; Rajalakshmi _e_ta_l_., 1980). Both MNU and DMN exposure 12 £332 has been shown to result in the formation of the following alkylation products: 7—methylguanine, O6- methylguanine, 3-methyladenine, l-methyladenine, and methylphophotri- esters (Lawley, 1973, 1974; Pegg and Nicoll, 1976; Bouchert and Webb, 1977; Pegg, 1977a) (Figure 3). It is the formation and persistence of "pro-mutagenic" methylated purines in DNA that has been proposed as one of the cellular modifications which could lead to tumor initiation following exposure to carcinogenic methylating agents (Lawley, 1974; Pegg, 1977a). Quantitatively, 7-methy1guanine is the major adduct formed follow- ing exposure to methylating agents and thus initially studies focused on it as the critical DNA adduct (Lawley and Brookes, 1961; Lawley, 1974). Lawley and Brookes (1961) proposed that since methylation at the N-7 position of guanine results in a dramatic increase in the rate of ionization at the N-l proton which is involved in Watson and Crick H-bonding, tautomers of guanine could mispair with thymine. The t% for 7-methy1guanine elimination in rat liver has been identified as approximately 70 hours and thus this base persists in DNA until undergoing spontaneous depurination and possibly a slow enzymatic removal (Margison g£_al,, 1973; Pegg, 1977b; Frei g£_al,, 1978; see Figure 13; Table 12). Since 7-methy1guanine persistance is similar for all organs of the rat (Pegg, 1977a), this fact could argue against enzymatic loss. Differences in the rate of chemical depurination 10 :> Adenine 5'2. 9"}> 603?:‘?. I3 7 m Figure 3. Major in_vivo sites of carcinogen-induced purine methylation. 11 occurring during incubation in gi££g_and influences of the nuclear environment on spontaneous depurination in 2322 might more accurately account for the slightly more rapid loss of 7-methy1guanine $332122 rather than speculation of enzymatic processes (Pegg, 1977a). Despite Lawley's suggestion, 7-methylguanine does not appear to be "pro~mutagenic" in_ziyg_as it does not lead to the mis-incorporation of nucleotides by nuclei acid polymerases (Ludlum, 1970). As well, the persistance of 7-methy1guanine in DNA has not been correlated to the carcinogenicity of N—nitroso compounds (Swann and Magee, 1968; Pegg and Nicoll, 1976; Pegg, 1977a). Methylmethanesulfonate produces more 7- methylguanine in DNA of rat kidney tissue than dimethylnitrosamine or methylnitrosourea yet it is not a renal carcinogen in rats whereas the other two chemicals are potent inducers of kidney tumors (Swann and Magee, 1968; 1971). Loveless in 1969 suggested that 06-methy1guanine was the most likely promutagenic adduct. Gerchman and Ludlum (1973) have studied the properties of 06—methy1guanine in templates for RNA polymerases and have substantiated the suggestions that 06-methylguanine is a "pro- mutagenic base". Gerchman and Ludlum (1973) found that UMP or AMP was substituted for GNP in transcriptional RNA products. 06—methy1guanine loss from DNA has been identified as an enzyme dependent as well as dose-dependent process (Kleihues and Margison, 1976; Pegg, l977a;b; Pegg and Hui, 1978a). 06—methylguanine is removed more rapidly from'rat liver DNA when doses of dimethylnitrosamine were less than 2.5 mg/kg as compared to larger doses 4 mg/kg up to 20 mg/kg (Pegg, 1977b). 12 Correlations between the formation and persistance of 06-methyl- guanine in DNA till replication and the subsequent tumor incidence in those same tissues has been variable (Den Engelse, 1974; Pegg and Nicoll, 1976; Pegg, l977b; Frei ggnal., 1978). Frei £5 El. (1978) have demonstrated that the carcinogenic effec- tiveness of methylnitrosourea, ethylnitrosourea and ethyl methanesul- phonate to produce thymic lymphoma was positively correlated with the extent and persistance of alkylation of DNA at the O6-guanine position in thymus tissue. Pegg and Nicoll (1976) have also shown a positive correlation between the formation and persistance of O6—a1ky1guanines and susceptibility to tumor incidences when they compared alkylation levels in liver and kidney tissues following exposure to dimethyl- nitrosamine, methylnitrosourea and diethylnitrosamine. However, when 06-methy1guanine persistance and subsequent liver carcinogenesis were compared in GR and C3Hf mice following DMN exposure, such a correlation did not exist (Den Engelse, 1974). Levels of 06-methylguanine were higher in the livers of the resistant GR mouse strain. Similar find- ings were reported by Buecheler and Kleihues (1977) when they compared 06-methylguanine persistance in brain DNA and susceptibility to neural tumor induction in mouse strains A/J and C3HeB/FeJ. C3HeB/FeJ mice which were especially susceptible to neural tumor development did not have larger initial levels of 06-methy1guanine nor did 06-methylguanine persist in brain tissue longer than resistant A/J mice strains. These findings suggest that the organotropic effects of alkylating carcinogens can not be explained solely on the extent and persistance of 06-methylguanine in DNA. 13 Less evidence concerning the possible biological implications of 1- and 3-methy1ations of adenine is present in the literature, but the possibility of mutation by 3-a1kyladenine has been suggested via AT + CO transitions (Lawley, 1974; Pegg, l977a). Removal of 3-methyladenine from DNA appears to be faster than solely spontaneous depurination and therefore enzymatic mechanisms were also proposed for this base (Margi— son and O'Connor, 1973; Shackleton 35 al., 1979). A 3-methyladenine DNA-glycosylase has been partially purified from human lymphocytes (Brent, 1979). The possibility of non-enzymatic depurination of methylated bases and substitution of alternative bases via error prone DNA repair has also been suggested as a possible mutagenic mechanism in eucaryotic cells (D'Ambrosio and Setlow, 1976; Sarasin and Hanawalt, 1978; Das Gupta and Summers, 1978). However, there is no demonstration of such an effect in the DNA of mammalian cells. Methylphosphotriesters persist in the DNA of rat and mice tissues following exposure to MNU or DMN demonstrating their chemical stabi- lity (Bannon and Verly, 1972; Lawley, 1974; Shooter £3 31., 1977). These studies also suggest that phosphotriesters are not subject to enzymatic repair. Their persistance in brain and liver has not been correlated with subsequent tumor formation (Rajewsky_g£flal., 1976). IV. Intragenomic Distribution of Carcinogen-induced Damage Not only must the question of what carcinogen—induced alkylations play a role in carcinogenesis be answered but also where in the chro- matin complex such alkylations occur. Identification of where l4 carcinogen damage is occurring in relationship to areas of chromatin which are template active (euchromatin) or template inactive (hetero— chromatin) may help to explain the events following carcinogen binding to DNA which could lead to tumor formation. Is DNA damage, which could result in initiation, occurring randomly in chromatin or are there mutational "hot spots"? It is relatively easy to understand the biological consequences of mutations in transcriptionally active regions of the genome but less conceivable to understand the phenotypic consequences of mutations in inactive genome regions. Promotion might result in shifts in gene expression that could result in phenotypic expression of previous unexpressed genotypic damage. Thus, investi- gations of intragenomic differences in methylation and of repair of these specific carcinogen-induced damage sites should prove valuable in understanding the process of carcinogenesis. Although some studies on the intragenomic distribution of carcinogens have been conducted, the question of whether or not carcinogen-induced alkylation is selective for specific regions of the genome remains to be definitively answered. Ramanathan g£_al, (1976a) designed a series of experiments to study if methylation of rat liver chromatin DNA by DMN and subsequent removal (iggziyg) of DNArbound methylated products was random or non- random. Comparisons were made between the methylation of nuclease sensitive and resistant regions of chromatin DNA. DNase I and micro- coccal nuclease were used to digest chromatin DNA until 50 percent was rendered acid soluble (nuclease sensitive fraction). Results from these experiments revealed that 70 percent of the methylated products were associated with the nuclease sensitive regions of chromatin DNA 4 hours after DM N exposure. This non-random distribution of alkylation 15 was demonstrated for both pancreatic DNase I and micrococcal nuclease digestion. Ramanathan g£_al, (1976b) have also shown that not only was the extent of methylation of nuclease accessible regions of DNA lower but as well, rates of removal were slower. Pegg and Hui (1978a), like Ramanathan §£_al, (1976b), have also investigated the intragenomic distribution of methylation products following DMN exposure. The experiments which Pegg and Hui designed were conducted to ascertain if 1) the efficient removal of 06-methylgu- anine after low doses of DMN could be due to preferential methylation of more readily repairable sites in chromatin, and 2) to determine if the loss of other methylated purines from rat liver DNA would show similar dose dependency and distribution within the chromatin as 06- methylguanine and thus implicate a shared excision system. The methods that Pegg and Hui (1978a) employed to prepare and digest liver chromatin were essentially identical to those utilized by Ramanathan 35 El! (1976b). The carcinogen DMN was administered i.p. as Ramanathan §£_§l, (1976), although female Sprague—Dawley rats were used by Pegg and Hui in contrast to the male Wistar rats used by Ramanathan g _a_1_. (1976b) . 06—Methylguanine was easily detected by Pegg and Hui (1978a) 4 hrs after 0.75 or 20 mg/kg injection and also 24 hrs after the higher dose of 20 mg/kg but was reduced substantially 24 hrs following the smaller dose. 0n the other hand, 7—methylguanine associated radioactivity showed minimal decline between 4 hrs and 24 hrs and a similar extent of decline was observed after both doses. Radioactivity was also found to be associated with alkylated pyrimidine nucleotides, phosphate alkyla- tion, l-methyladenine, 3-methyladenine, 3-methylguanine and l6 7-methyladenine. There appeared to be a substantial decrease in the radioactivity present in all of these bases between 4 to 24 hrs after administration of either dose (90% decrease for 7-methy1adenine and 50% decrease in 3-methylguanine). Thus, only 06-methylguanine demonstrated a definite relationship between initial extent of alkylation and effi- ciency of removal. Radioactivity associated with alkylated pyrmidines and phosphates declined only slightly during the 4 to 24 hr periods and the dose of DMN did not affect this decline. Pegg and Hui (1978a) investigated the possibility that alkylation of 06-methylguanine at low doses occurs preferentially at chromatin sites which are more accessible to excision repair. First, the distri— bution of 06-methylguanine was assessed in terms of nuclease sensitive vs. nuclease insensitive chromatin regions and second, the effect of large doses of unlabelled DMN on the persistance of radioactively labelled 06-methylguanine was investigated. The nuclease digestion (DNase I and micrococcal nuclease) that Pegg and Hui (1978a) used determined that approximately 50% of the total DNA was nuclease sensitive. This concurred with the studies done by Ramanathan gt_al, (1976b). However, Pegg and Hui's study showed that the release of radioactivity paralleled the release of acid soluble nucleotides. In no case was it greater than release of nucleo- tides. This is in opposition to Ramanathan's observations that release of radioactivity was greater. Pegg and Hui (1978a) speculate that allowance for radioactivity present in acid treated supernatant frac— tion from non-nuclease exposed tubes was not subtracted by Ramanathan g; al. (1976b). However, Ramanathan gt al. (1976b) specifically stated 17 that non-enzymatic background levels were subtracted from their values. These discrepancies have not been resolved. Pegg and Hui (19783) have examined nuclease sensitive and insensi- tive chromatin regions in terms of amount and persistance of methylated bases at 4 or 24 hrs after 3 different doses of DMN (25 ug/kg, 1.5 mg/kg and 20 mg/kg). The content of 7-methylguanine and O6- methylguanine was found to be the same in nuclease—sensitive and resistant fractions. The distribution of alkylated guanines did not differ with the three doses of DMN administered. Although significant loss of 06-methy1guanine occurred after 24 hours, this decrease occurred to the same extent in both nuclease-accessible and inaccessible chroma- tin regions. Pegg and Hui (1978a) have also investigated the effects of large doses of unlabelled DMN on 06-[C14]-methylguanine persistance. The large doses (20 mg/kg) of unlabelled DMN were given concurrently with a dose of CIADMN of 0.75 mg/kg and at 1 hr after the radioactive ClA—DMN injection. Pegg and Hui found that the rate of loss of 06-methy1gu- anine was the same as that observed when the high dose of unlabelled DMN was given at the same time as the radioactive DMN. These studies suggest that the removal of 06—methylguanine was only dependent on the total extent of alkylation rather than reaction with selective sites in DNA at low doses. Cooper and Itzhaki (1975) have demonstrated a heterogeneous distri- bution of DNA alkylation products in rat liver chromatin 3 hours follow- ing administration of CIA-dimethylnitrosamine. In these studies, male Wistar rats were given i.p. doses of 2 mg/kg body weight. Poly—L-lysine 18 binding to DNA was used to render DNA resistant to DNase I degradation. This procedure is based on the knowledge that approximately 45% of DNA phosphates are available to bind with large molecules such as poly-L- lysine. Due to poly-L-lysine binding to DNA only DNA in PL inacces- sible zones is degraded (50%). Chromatin was isolated 5 hrs after dimethylnitrosamine treatment. Following poly-L—lysine binding, nuclease digestion and radioactivity assessment, it was found that the amount of alkylation in DNA was lower in poly-L-lysine binding (nuclease insensitive) regions. This observation agrees with the studies con- ducted by Ramanathan 35 al. (1976b) where non-random alkylation of DNA was observed following DMN exposure. Galbraith gt al. (1979) has also looked at distribution of 7- methylguanine in chromatin DNA isolated from hepatic tissue of rats injected with [Ola-methyl] dimethylnitrosamine (2 mg/kg). The level of 7amethylguanine in DNase I degradable DNA was 1.3 times that of whole DNA. These studies also agree with Ramanathan ggugl. (1976b). Galbraith and Itzhaki (1978) have examined the distribution of 7- methylguanine in 3 kinetic classes of DNA; highly repetitive, middle repetitive and single copy sequences of DNA isolated from the livers of rats injected with Cl4-dimethylnitrosamine. The distribution of 7- methylguanine in these three kinetic classes was random. Rao fig 21- (1977) have examined the distribution of specific methylated bases following exposure to methylnitrosourea. These in- vestigators found after DNase I nuclease digestion of 50% of rat hepatic chromatin DNA that 40% of the 06-methylguanine, 55% of the 7- methylguanine and less than 20% of the 3-methyladenine content was acid soluble. (Note: These percentages are minus background non-enzymatic 19 base release values.) Thus, these studies indicate a non-random di- stribution of specific methylated bases following exposure to methyl- nitrosourea. An alternative approach to assessing the question of whether carcinogens interact with specific DNA regions has been to assess the effects of binding ligands on carcinogen—induced methylation of DNA (Kolchinskii g£.al., 1975; Rajalakshmi_g£mgl., 1978; Rajalakshmi 33 31., 1980). Rajalakshmi 35 31. (1980) have recently investigated the nature of DNA regions accessible to methylnitrosourea, methylmethanesulfonate and dimethylsulfate-induced methylation. To delineate the characteristics of DNA regions which were accessible, the extent of DNA methylation in the presence and absence of DNA binding ligands, spermidine and dista— mycin A were examined. Spermidine, a polycationic amine which binds strongly to DNA to neutralize DNA phosphate groups, inhibited 7- methylguanine formation inuyi££g_in rat liver DNA following MNU expo- sure but not methylmethanesulfonate (MMS) nor dimethylsulfate (DMS) exposure. Distamycin A, another DNA binding ligand which like spermine appears to stabilize the secondary structures of DNA associated with A—T rich regions, also inhibited DNA methylation by MNU but not MMS nor DMS. These studies suggest a biochemical basis for non—random alkyla- tion of DNA by demonstrating that alkylation of DNA by an "SNl" type carcinogen such as MNU can be inhibited by alterations in DNA con- formation and neutralization of charged DNA moieties. Rajalakshmi 35 .El- (1978) have shown the inhibition of MNU-induced DNA methylation by spermidine occurs not only for 7-methy1guanine but also for O6~methylgu- anine and 3-methyladenine. Kolchinskii g£.al. (1975) have shown that 20 dimethylsulfate induced 3~methyladenine formation in DNA bound with distamycin A was inhibited but 7-methylguanine formation was not. This was probably due to the location of the 3 position of adenine in the narrow groove where distamycin A was binding. In summary, the investigations cited above suggested that methyl- nitrosourea induced methylation occurs at guanines located near the binding sites of distamycin A or spermine whereas methylmethanesulfo- nate and dimethylsulfate induced guanine methylation occurs at sites far from the ligand binding sites. MNU induced alkylation at adenine and guanine was inhibited when DNA underwent confirmational changes occurring upon stereospecific ligand binding along the minor groove of DNA. Methylmethansulfonate and dimethylsulfate methylation of the N-7 position of guanine was unaffected. Therefore, it appears that SNl type carcinogens interact with different DNA regions than SN2 type carcinogens. Carcinogen binding to chromatin has also been assessed following exposure to polycyclic hydrocarbons and aromatic amines. 3-Methylcho— lanthrene and ],2,5,6—dibenzanthracene binding to DNA from AKR mouse embryo cells has been reported to occur preferentially in chromatin fractions which co-sediment with transcriptionally active chromatin through sucrose gradients (Moses g£_al,, 1976). The weaker carcinogen l,2,3,4-dibenzanthracene exhibited minimal binding to this chromatin fraction. 12 zitrg experiments have discerned preferential binding of N-acetoxy-N-Z-acetylaminofluorene binding to DNA in regions of chro- matin sensitive to staphylococcal nuclease (Metzger and Daune, 1976). 21 Ig_giyg studies distinguishing regions of chromatin DNA on the basis of their sensitivity to DNase I have demonstrated the increased initial binding of N-hydroxy—Z-acetylaminofluorene to DNase I resistant regions (Ramanathan g£_al,, 1976a; Metzger_g£-al., 1977). Moyer g£_§l, (1977) have studied the ig_zi!g_binding of N—hydroxy-N-Z-acetylaminofluorene to rat liver DNA separated into apparent hetero- and euchromatin fractions by differential sedimentation through a sucrose gradient. They observed greater amounts of carcinogen bound to euchromatin than heterochromatin fractions and the rates of loss of bound carcinogen were greater for the euchromatic fractions. Using both a glycerol gradient chromatin fractionation procedure and a selective MgCl2 chromatin precipitation procedure, the initial binding of N-hydroxy-Z- acetylaminofluorene was also observed to be greater on euchromatin DNA (Schwartz and Goodman, 1979b). In addition, Schwartz and Goodman (1979c) have shown that non-random nature of 2-acetylaminof1uorene- induced alterations of DNA template capacity including possible func- tionally significant carcinogen-induced modifications of DNA-adenine. Although carcinogen binding to chromatin DNA appears to be non- random for carcinogens such as polycyclic hydrocarbons and aromatic amines the situation appears to be less well defined for methylating agents such as methylnitrosourea and dimethylnitrosamine. Although various methods of chromatin fractionation have been utilized (glycerol and sucrose gradient centrifugation, DNase II and micrococcal nuclease sensitivity, MgCl2 precipitation, etc.) to determine the intragenomic distribution of carcinogen-induced methylation, it is not yet clear whether the binding of such agents is non—random. 22 V. Chromatin Fractionation The studies reported in this thesis have utilized an alternative method of chromatin fractionation, the DNase II-MgCl2 precipitation method of Gottesfeld ggngl. (1974) to separate fractions of chromatin having euchromatic characteristics (template active, 82 fraction) from areas of the genome having heterochromatic characteristics (template inactive, P2 fraction) (Figure 4). Evidence indicating that the DNase II-IMgCl2 fractionation procedures is valid has been published by numerous investigators (Gottesfeld SE al., 1974; Gottesfeld g£_al,, 1976; Matsui and Busch, 1977; Hendrick ggngl., 1977; Hemminki and Vauhkonen, 1977; Samuel 2£H§1., 1977). Gottesfeld g£_gl, (1974) have examined rat liver chromatin frac- tionated by the DNase II-MgCl2 method. The $2 fraction was enriched in non-histone proteins, depleted in histone proteins and was also shown to be 3 times superior than unfractionated chromatin in template activity for RNA synthesis. A 6-7-fold enrichment in template-active sequences was observed in the 32 fractions. Gottesfeld 35 El. (1976) have shown that DNA isolated from the 82 fraction of rat liver chromatin contains a subset of nonrepetitive DNA sequences and a family of middle repetitive sequences. The $2 fraction has also been identified as enriched in nonrepeated sequences which are transcribed in yizg_into cellular RNA. When DNA sequences in the $2 fraction of brain tissue were compared with the sequences in liver 82 DNA, a low proportion of sequences in common was observed. 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Amounts of the four methylated bases 7-MeG, 06—MeG, l-MeA and 3-MeA in total chromatin and heterochromatin (P2) were assessed by HPLC separation and radioactive scintillation counting. A recovery of 99(:2.0)% of the radioactivity put onto the HPLC column was observed. The values in this table represent the mean : S.E. of the data obtained from three individual determinations. 2Total chromatin DNA alkylation represented 0.903(:0.2)x10-3 umoles methyl/mg DNA. The purine hydrolysate from total chro- matin represented 74(:l6)% of the total chromatin DNA alkylation. 3Tot 1 heterochromatin DNA alkylation represented 1.12(:0.1l) x 10 umoles methyl/mg DNA. The purine hydrolysate from hetero- chromatin represented 60(:l.8)% of the total heterochromatin DNA alkylation. 79 .mdoHumaHaumuow ucmvdmmmv In“ mounu How .9.m H some mam ucmmmumou monam> mmonH .cowumazxam <29 aHumaounooumumn com afiumaouno.amuou mnu mo Naa.¢uvmm mam NAw.vamw wmucmmmummu mummkaouw%£ ofiafiudm one .vmum>oomu mos canaoo 0999 mnu ouco usm %ufi>wuomoavmu mnu mo NAH.NHme .mu9 u: «N Am ooa\maoan\won OH .mmawan mHv Dzzwmm ou.wmm09xm mums Eoum wmumaomfi mums mcofiuomum cfiumaouso .Dzzwmm cu muomomxm waHBoaaom <29 cwuma lounoououmn msmum> cfiumaouno Hmuou ow momma wmumaznuma owmwommm mo aowuma%xa< .wH musmfim 80 TC 7-M.G 04m l-MeA 3-M.A 7-MoG 06-1“; l-MoA 3-M.A L__l__|_\ I o o o $ 9 o o 52 CD It) co cu '- ' (gouwNo Bw/Ihuaw 5mm d %~_ P2 18 Figure 81 TABLE 11 Purine Methylation in DNA from Total Chromatin and Heterochromatin Following Exposure to 3H-MNUl pmoles Methyl (x106) Z of Total DNA Fraction Base mg DNA Alkylation Total Chromatin géMeG 89.6 :10.5 69.4 :5.12 O éMeG 1.16 : 0.12 0.92:0.16 1-MeA 0.667 : 0.11 0.53:0.11 3+MeA 1.67 : 0.11 1.31:0.12 Heterochromatin g-MeG 105 :11.8 71.2 :5.13 O -MeG 1.24 : 0.076 0.86:0.13 léMeA 0.739: 0.102 0.50:0.039 3-MeA 1.38 : 0.38 1.01:0.34 lChromatin fractions were isolated from rats exposed to 3H—MNU (15 pmoles, 1.0 uCi/umole/lOO g) 24 hr post—intubation. Amounts of the four methylated bases 7-MeG, 06—MeG, l-MeA and 3-MeA in total chromatin and heterochromatin (P2) were assessed by HPLC separation and radioactive scintillation counting. A recovery of 86(:2)% of the radioactivity put onto the HPLC column was observed. The values in this table represent the mean : S.E. of the data obtained from three individual determinations. 2Total chromatin DNA alkylation represented 1.3(:0.18) x 10'”4 umoles methyl/mg DNA. The purine hydrolysate from total chro- matin represented 82(:9)Z of the total chromatin DNA alkylation. 3The purine hydrolysate from heterochromatin represented 75(:4)% of the total heterochromatin DNA alkylation. Tot l heterochro- matin DNA alkylation represented l.5(:0.24) x 10 umoles methyl/ mg DNA. 82 Figure 19. Comparison of MNU- and DMN—induced patterns of purine alkylation. Chromatin fractions were isolated from the livers of rats 24 hr after exposure to 3H-MNU (15 pmoles, 10 uCi/umole/100 g) or 4C—DMN (8 pmoles, 1.0 uCi/umole/lOO g). Assessments of 7-MeG, O -MeG, l—MeA and 3+MeA content in heterochromatin (P2) and total chromatin DNA were made. Following MNU exposure, total chromatin and hzterochromatin DNA alkylation equaled l.3(:0.l8)x10'4 and 1.5(:0.24)x10- umoles methyl/mg DNA, respectively. The 74MeG content of total chromatin represented 69(:5.l)% of total chromatin DNA alkylation. The 7-MeG content of heterochromatin equaled 71(:5)% of total heterochromatin DNA alkylation. Following DMN exposure, total chromatin and he erochromatin DNA alkylation equaled 0.903(:0.2)x10'3 and 1.12(:0.1l)x10 umoles methyl/mg DNA, respectively. The 7-MeG content of total chromatin re- presented 62(:13)% of total chromatin DNA alkylation. In heterochromatin 7—MeG content represented 56(:2)% of total heterochromatin DNA alkylation following DMN exposure. Values represent mean : S.E. for three rats. 4 Rollo lo 7-MeG 83 0.07 0.01 *- fill dime I-MeA 343.1; - 06m I-MEA 3 mm P2 DMN MNU 0.07 *- 005- . I i I , - ohms l-Mo 3-MeA came l-MaA 3-MeA TC TC ‘ DMN MNU Figure 19 84 these experiments have been expressed as a ratio of the methylated bases to 7—MeG. Comparisons can be made between P2 and total chro— matin DNA alkylation at 24 hr following DMN or MNU exposure. No statistical differences could be ascertained in the pattern of purine alkylation of total chromatin versus heterochromatin following expo- sure to either carcinogen. At 24 hr following MNU exposure, Figure 19 shows that the ratio of 06-MeG/7—MeG was 0.012 for both total chromatin and heterochroma- tin. The léMeA/7-MeG ratio in total chromatin and heterochromatin were 0.0075 and 0.0070, respectively. The 3-MeA/7—MeG ratio in total chromatin and heterochromatin at 24 hr following MNU exposure equalled 0.019 and 0.014, respectively. At 24 hr following DMN exposure, Figure 19 shows that the ratio of 1-MeA/7-MeG in total chromatin was 0.11 and was equal to the ratio obServed in heterochromatin. The 064MeG/7-MeG ratios in total chro- matin and heterochromatin following DMN exposure were 0.063 and 0.061, respectively. The 3—MeA/7—MeG ratios were 0.022 and 0.018, respec- tively for total chromatin and heterochromatin at 24 hr following DMN exposure. Specific methylated purines have also been assessed at 3 hr post— MNU exposure. Figure 20 compares methylated purines in total chroma- tin versus heterochromatin. Total chromatin DNA alkylation repre- sented 1.25x10-4 umoles methyl/mg DNA. Total heterochromatin DNA alkylation represented 1.18x10-4 umoles methyl/mg DNA. 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