As noted above, the majority of chemical carcinogens must be metabolized within the cell be- fore they exert their carcinogenic activity. In this respect, metabolism of some chemicals results in a bioactivation instead of elimination. Thus, metabolic capabilities may underlie how a sub- stance that is not carcinogenic for one species may be carcinogenic for another. This becomes important for carcinogen testing in whole animals for both hazard identification and risk assess- ment. Such considerations impact directly on the choice of the most sensitive species or the spe- cies most similar to humans for these evaluations.
Since chemical carcinogens are reactive per se or are activated by metabolism to reactive intermediates that bind to cellular components including DNA, their electrophilic derivatives— which bound to a variety of nucleophilic (electron-dense) moieties in DNA, RNA, and protein— were considered the ultimate carcinogenic form of the compounds of interest (see above). Sev- eral lines of evidence indicate that DNA is the critical target for carcinogenesis. The first hint that DNA was the target for heritable alterations due to carcinogen administration was from the increased incidence of cancer in genetically prone individuals with defective ability to repair DNA damage—e.g., xeroderma pigmentosum, Bloom syndrome, Cockayne syndrome, etc. (Smith, 1991). The second major piece of evidence that DNA was the target of carcinogen action was the observation of carcinogen-induced mutations in specific target genes associated with neoplasia in a multitude of experimental systems. A comparison of DNA adduct formation with biologically effective doses of carcinogens with different potencies demonstrated that the level of DNA damage was relatively similar. Since covalent adducts in DNA could be derived from carcinogenic compounds, the mechanism by which mutations arise and their relationship to carcinogenesis was the next area to be examined in the quest for an understanding of cancer development.
The induction of mutations is due primarily to chemical or physical alterations in the structure of DNA that result in inaccurate replication of that region of the genome. The process of mutagenesis consists of structural DNA alteration, cell proliferation that fixes the DNA dam- age, and DNA repair that either directly repairs the alkylated base(s) or results in removal of larger segments of the DNA (cf. Naegeli, 1994). Electrophilic compounds can interact with the ring nitrogens, exocyclic amino groups, carbonyl oxygens, and the phosphodiester backbone of DNA (Chang et al., 1994). Carcinogenic agents that result in formation of bulky adducts often
specifically react with sites in the purine ring. For example, aromatic amines bind to the C8 po- sition of guanine, while the diol epoxide of polycyclic aromatic hydrocarbons binds to the N2 and N6 position of guanine (see above). The position of an adduct in DNA and its chemical and physical properties in that context dictate the type(s) of mutations induced (Essigmann and Wood, 1993). This indicates that different adducts can induce a distinct spectrum of mutations and additionally that any given adduct can result in a multitude of different DNA lesions. Con- firming the need for metabolic activation of carcinogenic compounds to their ultimate reactive form it was demonstrated that, whereas 2-acetylaminofluorene itself is not mutagenic, its sulfate metabolite was highly mutagenic for transforming DNA (Maher et al., 1968). These findings led to the development of mutagenesis assays for the detection of chemical carcinogens from the premise that one could detect carcinogens in highly mutable strains of bacteria given exogenous liver microsomal preparations for in vitro metabolism of the test agent (Chapter 13). Cultured mammalian cells have also been developed for evaluation of the mutagenic action of potential carcinogenic agents. Compounds are evaluated in the presence (Michalopoulos et al., 1981) or absence (Li et al., 1991) of metabolic activation systems such as irradiated hepatic feeder layers or hepatic microsomes. The use of these in vitro screens of mutagenicity has permitted analysis of the mutational specificity of some carcinogens (Table 3.3). While the data shown in Table 3.3 were derived from bacterial mutagenesis studies, several other systems have also been utilized in attempts to determine mutagenic specificity of various agents (Essigmann and Wood, 1993).
Point mutations, frameshift mutations, chromosomal aberrations, aneuploidy, and poly- ploidization can be induced by chemicals with varying degrees of specificity that are, in part, dose-dependent. Mutagenesis can be the result of several different alterations in the physical and chemical nature of DNA. While alkylation of DNA with small alkyl groups or large bulky ad- ducts can result in mutation, other processes may also be involved. Conformation of the DNA has a major impact on the potential mutagenic activity of a compound. This is best demonstrated by the related compounds 2-acetylaminofluorene and 2-aminofluorene, which both form bulky DNA adducts at guanine residues in DNA. The AAF adduct distorts the double helix, while the AF adduct remains outside the helix and does not distort the helix. The AAF adduct induces frameshift mutations, whereas that of AF induces primarily transversions (Bichara and Fuchs,
1985). Planar agents that can intercalate between the base pairs in DNA can effectively induce frameshift mutations by exacerbating slippage mispairing in repetitive sequences. In addition, agents that lie within the major or minor groove of DNA can perturb nucleosome formation and may alter DNA replication. Some of these agents are potential chemotherapeutic agents. Agents such as irradiation and topoisomerase inhibitors that induce double-strand breaks can also en- hance mutagenesis (Eastman and Barry, 1992).
Several mechanisms of mutagenesis exist. The presence of certain alkylation products, such as the O-6 alkyl deoxyguanosine and the O-4 alkyl deoxythymidine, permits a degenerate
base pairing able to base pair with the appropriate base as well as an inappropriate base. This can be demonstrated in vitro and in vivo as the induction of transition mutations after treatment with certain alkylating agents (Singer, 1986). Thus, methylating or ethylating agents result in muta- tions as a result of base mispairing. The active metabolites of compounds, such as polycyclic aromatic hydrocarbons and aromatic amines, form bulky DNA adducts that block DNA synthe- sis, resulting in a noncoding lesion. The synthetic machinery employs bypass synthesis to avoid the lethal impact of these unrepaired lesions (Friedberg, 1994). Under this condition, the most prevalent base, frequently deoxyadenosine (Shearman and Loeb, 1979), is inserted opposite the offending adducted nucleotide base. Thus, DNA binding and repair, induction of point muta- tions, and clastogenicity have proven useful as endpoints in the identification of potential carcin- ogens as well as biomarkers of carcinogen exposure (Chapter 13). The role of DNA repair in protection of the genome and in the induction of mutations is an essential component in the mu- tagenesis process (see below).
Not all chemical carcinogens require intracellular metabolism to become ultimate carcino- gens. Examples of direct-acting mutagens include alkylating agents such as β-propiolactone, nitrogen mustard, ethyleneimine, and bis(chloromethyl)ether (Figure 3.3). Direct-acting carcin- ogens are typically carcinogenic at multiple sites and in all species examined. A number of the direct-acting alkylating agents, including some used in chemotherapy, are carcinogenic for hu- mans (Chapter 11).