Knowledge of the metabolic activation of chemicals has dramatically advanced our understand- ing of carcinogenic mechanisms underlying the extreme diversity of chemical structures in- volved in cancer development. The relationship of chemical structure to carcinogenic activity plays a significant role in the potential identification and mechanism of potential chemical car- cinogens. Computerized databases of carcinogenic and noncarcinogenic chemicals have been developed to relate structure to carcinogenic activity in a variety of carcinogens (Enslein et al.,1994; Rosenkranz and Klopman, 1994).
Using as a primary basis the results of rodent bioassays on more than 500 chemicals, Ashby and Paton (1993) have studied the influence of chemical structure on both the extent and the target tissue specificity of carcinogenesis for these chemicals. From analysis of the presence of potential electrophilic sites (DNA reactive), mutagenicity to Salmonella, and level of carcino- genicity to rodents (Chapter 13), these authors have developed a list of chemical structures that possess a high correlation with the development of neoplasia in rodent tests (Ashby et al., 1989; Tennant and Ashby, 1991). These “structural alerts” signify that a chemical having such struc- tures should be examined closely for carcinogenic potential. These authors have developed a composite model structure indicating the various structural alerts that appear to be associated with DNA reactivity or carcinogenicity (Figure 3.11). The substantial database used to generate these structural alerts indicates the utility of this information for the identification of potential carcinogens and their mechanisms of their action in specific tissues. In addition, investigation of the metabolic activation of such functional groups during the carcinogenic process should pro- vide insight into their role in the induction of cancer.
Figure 3.11 The substituents are as follows: (a) alkyl esters of either phosphonic or sulfonic acids; (b) aromatic nitro groups; (c) aromatic azo groups, not per se, but by virtue of their possible reduction to an aromatic amine; (d) aromatic ring, N-oxides; (e) aromatic mono- and dialkylamino groups; (f) alkyl hydra- zines; (g) alkyl aldehydes; (h) N-methylol derivatives; (i) monohaloalkenes; (j) a large family of N and S mustards (ß-haloethyl); (k) N-chloramines (see below); (l) propiolactones and propiosultones; (m) aromatic and aliphatic aziridinyl derivatives; (n) both aromatic and aliphatic substituted primary alkyl halides; (o) derivatives of urethane (carbamates); (p) alkyl-N-nitrosamines; (q) aromatic amines, their N-hydroxy derivatives and the derived esters; (r) aliphatic and aromatic epoxides. The N-chloramine substructure (k) has not yet been associated with carcinogenicity, but potent genotoxic activity has been reported for it (discussed in Ashby et al., 1989). Michael-reactive a,ß-unsaturated esters, amides or nitriles form a rela- tively new class of genotoxin (e.g., acrylamide). However, the structural requirements for genotoxicity have yet to be established, and this structural unit is not shown in the figure. (Adapted from Tennant and Ashby,1991, with permission of the authors and publisher.)
DNA, RNA, AND PROTEIN ADDUCTS RESULTING FROM THEIR REACTION WITH ULTIMATE CARCINOGENIC FORMS
One of the most intriguing problems that experimental oncologists have considered in the area of chemical carcinogenesis is the characterization of the covalent compounds resulting from re- actions between the ultimate metabolite of a chemical carcinogen and a macromolecule. The structures of several different chemical carcinogens covalently bound or adducted to protein and nucleic acids are shown in Figure 3.12. For the detailed chemistry of the reactions involved in the formation of such adducts, students are referred to several of the references, especially those by the Millers (1970, 1978), Weisburger and Williams (1982), Hathway and Kolar (1980), and Dipple et al. (1985). Guanine is the nucleic acid base that has been found to react most avidly with the “ultimate” forms of chemical carcinogens. As noted in Figure 3.12, the reaction of the ultimate form of N-methyl-4-aminoazobenzene with polypeptides involves a demethylation of
Figure 3.12 Structures of some protein- and nucleic acid–bound forms of certain chemical carcinogens. The macromolecular linkages are shown schematically. Esters of 2-acetylaminofluorene react predomi- nantly with the 8-position of guanine, whereas the epoxide of aflatoxin B1 reacts primarily with the N-7 position of guanine. The ethano-adenine and ethanocytosine adducts result from the reaction of DNA with halogenated acetaldehydes or ultimate forms of vinyl chloride and related structures. 7,-(2-hydroxy- ethyl)guanine is a product of the reaction of ethylene oxide with DNA.
methionine and reaction of the electrophilic position ortho to the amino group of the azobenzene with the nucleophilic sulfur of methionine and subsequent loss of the methyl of methionine. Ad- ducts formed with DNA, which in most instances are similar to those seen in RNA, exhibit ste- reospecific configurations as exemplified by the reaction of the epoxide of aflatoxin B1 with N-7 position of guanine. AAF also reacts with guanine at the two positions of the DNA base, as
shown. The formation of an additional ring structure in adenine and cytosine occurs with the ultimate form of the carcinogen vinyl chloride as well as related chemicals that exhibit the same ultimate form (Bolt, 1988). In contrast, ethylene oxide directly alkylates the N-7 position of guanine in DNA (Bolt et al., 1988). An interesting adduction occurs during the metabolism of 2-nitropropane that causes the formation of 8-aminoguanine, possibly from the spontaneous re- action with the highly reactive intermediate (NH2+) that is probably formed during the metabo- lism of the nitro group (Sodum et al., 1993).
Of the number of chemical carcinogens that adduct DNA by direct methylation, ethyla- tion, or higher alkylations, several such agents are of considerable experimental and environ- mental significance. The positions alkylated by ethylating and methylating chemicals are noted in Figure 3.13, taken from the review by Pegg (1984). In this same discussion, Pegg described the relative proportions of methylated bases present in DNA after reaction with carcinogen- methylating agents (Table 3.1). The predominant adduct seen with methylating agents such as methylmethane sulfonate administered in vivo or in vitro is 7-methylguanine. In contrast, ethyla- tion of DNA is predominantly in the phosphate backbone. Pegg has argued that the principal carcinogenic adduct is the O6-alkylguanine. In contrast, Swenberg et al. (1984) reported that O4-alkylthymine may be a more important adduct for carcinogenesis because it remains in the DNA for much more extended periods than the O6-alkylguanine. The importance of the persistence of DNA adducts of ultimate carcinogens is discussed below.
Another common structural change in DNA is the hydroxylation of DNA bases. Such changes have been found in all four of the bases making up DNA (Marnett and Burcham, 1993), but the more commonly analyzed are 5-hydroxymethylthymine (Srinivasan and Glauert, 1990) and 8-hydroxyguanine (cf. Floyd, 1990). These hydroxylated bases have been found in DNA of target organs in animals exposed to chemical carcinogens, but they are also present in the DNA of organisms not subjected to any known carcinogenic agent (Marnett and Burcham, 1993). Es- timates of a rate of endogenous depurination of DNA of 580 bases per hour per cell and DNA strand breaks at a rate of 2300 per hour per cell have been reported (Shapiro, 1981). These esti-mates are not incompatible with the presence of oxidative DNA lesions at a level of 106 per cell in the young rat and almost twice this in the old rat (cf. Table 3.6; Ames et al., 1993). Such oxidative damage is presumably due to free radical reactions occurring endogenously in the cell that are capable of producing activated oxygen radicals (cf. Floyd, 1990; Ames et al., 1993). Such oxidative reactions, occurring either as a result of an endogenous oxidative phenomenon or from the administration of exogenous chemical and radiation carcinogens, are presumably rapidly repaired by mechanisms discussed below. Thus, endogenous mutations are kept to a minimum.
Finally, structural changes in DNA of largely unknown character have been reported through the use of a unique technology known as 32P-postlabeling (Reddy and Randerath, 1987). The outline of procedures used in this technology can be seen in Figure 3.14. After digestion of DNA to its constituent nucleotides, each nucleotide is labeled by using g32PO4-labeled ATP and a bacterial kinase, an enzyme that transfers the terminal phosphate of ATP to the available 5′ hydroxyl of the 3′ nucleotides to convert all of the nucleotides to a radioactive, biphosphorylated form. Nucleotides of the normal DNA bases are removed by appropriate chromatographic pro- cedures, leaving only those nucleotides that contain structural adducts. Although this technique has been used to demonstrate adduction of DNA by a variety of known chemical carcinogens, equally if not more interesting is the fact that a number of adducts of unknown structure have
Figure 3.13 Sites of alkylation of DNA under physiological conditions. (From Pegg, 1984, with permis- sion of the author and publisher.)
been discovered in living cells. Some of these structurally unknown DNA adducts, termed I-compounds (Li and Randerath, 1992), change with dietary modifications, drug administration (Randerath et al., 1992), and species and tissue differences (Li et al., 1990). I-compounds occur in human fetal tissues (Hansen et al., 1993), tend to increase with age and caloric restriction, but decrease in the liver during hepatocarcinogenesis (Randerath et al., 1991). Thus, the exact role if any of many DNA adducts of unknown structure in the process of carcinogenesis remains a question.
The mechanisms of inorganic chemical carcinogenesis have not been as well defined as those of organic chemicals. The interesting uniqueness of arsenic as a human carcinogen has not
Table 3.1 Relative Proportions of Methylated Bases Present in DNA After Reaction with Carcinogenic Alkylating Agents
yet been adequately explained. On the other hand, chromate induces DNA crosslinks in vivo in possible association with active oxygen radicals (Tsapakos et al., 1981; Costa, 1991). While the mechanisms of cadmium carcinogenesis are also relatively unclear, nickel as a carcinogen both in animals and humans apparently may effect carcinogenesis by a variety of mechanisms, in- cluding alterations in the structure of DNA itself (Sunderman, 1989; Costa, 1991).
The best-known and structurally identified normal modification of DNA is the methyla- tion of deoxycytidine residues by the transfer of a methyl group from S-adenosylmethionine by DNA methyltransferase (Holliday, 1989; Michalowsky and Jones, 1989). Such methylation re- sults in the heritable expression or repression of specific genes in eukaryotic cells. When such methylation occurs during development, the expression or repression of specific genes may be “imprinted” by DNA methylation at various stages during development (Chapter 5; Barlow,1993). Chemical carcinogens may inhibit this process by several mechanisms, including the for- mation of covalent adducts, single-strand breaks in the DNA, and the direct inactivation of the enzyme DNA S-adenosylmethionine methyltransferase, which is responsible for normal methy- lation (cf. Riggs and Jones, 1983). Therefore the inhibition of DNA methylation by chemical carcinogens may represent a further potential mechanism for carcinogenesis induced by chemi- cals. That such a mechanism may be important in hepatocarcinogenesis was reported by Mikol et al. (1983), wherein half of the animals receiving a defined diet devoid of methionine and cho- line developed hepatocellular carcinomas and cholangiomas when subjected to this regimen for
18 months. The methyl-deficient diet induces a drastic hypomethylation of hepatic nuclear DNA (Wilson et al., 1984).
From this brief survey, the student may appreciate that the role of structural adducts of DNA in carcinogenesis is not a simple matter of adduct = mutation = carcinogenesis. Defined adducts of known complete chemical carcinogens, as exemplified by those depicted in Figure 3.11, generally may be considered to have a significant role in carcinogenesis induced by their procarcinogenic forms. However, the role and function of structurally undefined adducts such as I-compounds in the carcinogenic process is not so clear, nor is there substantial evidence that
Figure 3.14 The basic features of 32P-postlabeling assay for carcinogen-adducted DNA. The 32P-assay involves four steps: digestion of DNA, 32P-labeling of the digestion products, removal of 32P-labeled non- adduct components, and thin layer chromatography mapping of the [32P] adducts. Asterisks indicate the position of the 32P-label. (Modified from Gupta et al., 1982.)
such adduction leads directly to mutation. Finally, the inhibition of normal methylation of DNA may itself play a role, as yet undefined, in the carcinogenic process.