The extent to which DNA adducts occur as the result of the administration of chemical carcino- gens depends on the overall metabolism of the chemical agent as well as the chemical reactivity of the ultimate metabolite. Once the adduct is formed, its continued presence in the DNA of the cell depends primarily on the ability of the cellular machinery to repair the structural alteration in the DNA, the mechanisms for which are discussed below.
Persistence of DNA Adducts
It is from many of the considerations noted above as well as the presumed critical nature of the adduct in the carcinogenic process that a working hypothesis has evolved which postulates that the extent of the formation of DNA adducts and their persistence in the DNA should correlate with the biological effect of the agent (Neumann, 1983). In accord with this hypothesis, several studies have correlated the persistence of DNA adducts occurring during chemical carcinogene- sis with the high incidence of neoplasms in specific tissues (Table 3.2). Among the earliest of these studies was that of Goth and Rajewsky (1974), who demonstrated the relative persistence
of O6 ethylguanine in DNA of brain but not liver of animals administered ethylnitrosourea at 10
days of age. The rapid loss of the adduct in liver DNA contrasted with the seven times slower loss in DNA of the brain correlated with the appearance of neoplastic lesions in these tissues later in life. Swenberg and associates (1985) demonstrated an analogous situation in the liver, wherein administration of symmetrical dimethylhydrazine induced a high incidence of neo- plasms of hepatic vascular endothelium but a very low incidence in parenchymal cells of this
tissue. Examination of the same adduct, O6-ethylguanine, demonstrated its rapid removal from
the DNA of hepatocytes but much slower removal from the DNA of nonparenchymal cells in the liver, a large proportion of which are vascular endothelial cells. Similarly, Kadlubar and associ- ates (1981) demonstrated that more guanine adducts of 2-naphthylamine persisted in bladder epithelium (urothelium) than in liver after administration of the carcinogen to dogs. The bladder, but not the liver, is a target for this carcinogen and, as discussed earlier, the metabolic activation of this chemical and other aromatic amines appears to be different in the two tissues (see above). When the susceptibility to carcinogenesis by diethylnitrosamine was investigated in the same tissue in two different species, only the DNA of hamster lung exhibited significant alkylation and the development of neoplasia.
While the correlations noted in Table 3.2 support the working hypothesis of the impor- tance of specific adducts during the carcinogenic process, this is not the entire picture. Swenberg et al. (1984) demonstrated that the O4-ethylthymine adduct but not the O6-ethylguanine adduct is stable in liver parenchymal cells after the continuous exposure of rats to diethylnitrosamine. Fur- thermore, Müller and Rajewsky (1983) found that the O4-ethylthymine adduct persisted in all organs after the administration of ethylnitrosourea to neonatal or adult rats. Later studies by Ra- jewsky and associates (1998) found that the O6-ethylguanine adduct was removed from the DNA of specific genes in the mammary gland some 20 times faster than the O6-methylguanine adduct. Persistence of DNA adducts of the carcinogenic trans-4-aminostilbene did not correlate with tissue susceptibility. While the liver and kidney exhibited the greatest burden and persis- tence of the adduct, and the ear duct glands of Zymbal the least adduct concentration, it is the latter tissue that is most susceptible to carcinogenesis by this agent (Neumann, 1983).
Despite these and other exceptions to the working hypothesis, our knowledge of the per- sistence of covalent adducts of DNA and carcinogenic chemicals in tissues has been utilized in attempts to quantitate the exposure of humans to carcinogenic chemicals and relate the potential risk of neoplastic development to such exposure. The occurrence of adducts of benzo(a)pyrene throughout the tissues of exposed animals at unexpectedly similar levels (Stowers and Anderson,
1985) further supports the rationale for the investigation of persistent DNA adducts as well as carcinogen-protein adducts in the human. Immunological and highly sensitive chromatographic technologies have been used to demonstrate the presence of persistent DNA adducts of several carcinogenic species (Perera et al., 1991; Shields and Harris, 1991). The detection of DNA ad- ducts of carcinogenic polycyclic aromatic hydrocarbons has been demonstrated at relatively high levels in tissues, especially in blood cells of smokers and foundry workers, compared with nonexposed individuals (Perera et al., 1991). Huh et al. (1989) have demonstrated an increased
level of O4-ethylthymine in the DNA of liver from individuals with and without malignant neo-
DNA Repair, Cell Replication, and Chemical Carcinogenesis
The persistence of DNA adducts in relation to the development of neoplasia in specific tissues (Table 3.2) and the differences in the repair of the adducts are critical factors in chemical car- cinogenesis. The removal of methyl, ethyl, and similar small alkyl radicals from individual bases
Figure 3.18 Combinational specificities of heterocomplexes of gene products of mismatch repair genes. (a) Base/base mispairs; (b) insertion/deletion mispairs; (c) 5′ tailed DNA structures generated by single- strand DNA annealing following recombination—e.g., HR; and (d) Holliday junctions. (Adapted from Na- kagawa et al., 1999, with permission of authors and publishers.)
is to a great extent dependent on the presence of alkyltransferases (see above). While in some tissues, such as liver, it may be possible to increase the level of such enzymes in response to damage or hormonal or other influences, many tissues do not have an inducible repair mecha- nism. Furthermore, some adducts are extremely difficult if not impossible for the cell to repair.
One example of a lesion, the 3-(deoxyguanosine-N2-yl)-acetylaminofluorene adduct first de-scribed by Kriek and his associates (Westra et al., 1976), is depicted in Figure 3.12. This may in part account for the relatively wide spectrum of neoplasms inducible by this chemical carcinogen.
Of equal importance is the continuous damage to DNA that occurs within cells as a result of ambient mutagens, radiation, and endogenous processes including oxidation, methylation, deamination, and depurination. DNA damage induced by oxidative reactions (oxidative stress) is probably the source of most endogenous DNA damage. Ames et al. (1993) have estimated that the individual reactive “hits” in DNA per cell per day are of the order of 105 in the rat and 104 in
the human as a result of endogenous oxidative reaction. Such reactions can produce alkylation through peroxidative reactions such as those described in Figure 3.7 or hydroxylation of bases and single-strand breaks (Figure 3.15). The end product of oxidative damage to DNA can also be interstrand crosslinks and double-strand breaks (cf. Demple and Harrison, 1994) with the poten- tial for subsequent major genetic damage, as noted below. A more complete listing of the esti- mates of endogenous DNA damage and repair processes in the human is seen in Table 3.6. The data of this table emphasize the considerable degree and significant variation in types of DNA damage and repair that occur within each cell of the organism at a molecular level.
Experimental studies in mammalian cells have demonstrated that active oxygen radicals may contribute to clastogenesis directly (Ochi and Kaneko, 1989) and indirectly through the production of lipid peroxides (Emerit et al., 1991). While methods for the repair of some types of oxidative damage—including base hydroxylation (Bessho et al., 1993) and single-strand breaks (Satoh and Lindahl, 1994)—do exist, such repair requires time and may be dependent on many other intracellular factors. Since the formation of a mutation occurs during the synthesis of a new DNA strand by use of the damaged template, cell replication becomes an important factor in the “fixation” of a mutation. The importance of the rate of cell division and DNA synthesis in carcinogenesis has been emphasized by several authors (Ames et al., 1993; Butterworth, 1991; Cohen and Ellwein, 1991). Thus, while many DNA repair mechanisms themselves may not be abnormal in neoplastic cells compared with their normal counterpart, a high rate of cell divi- sion will tend to enhance both the spontaneous and induced level of mutation through the chance inability of a cell to repair damage prior to DNA synthesis. An important pathway of DNA repair that is genetically defective in a number of hereditary and spontaneous neoplasms in the human (Umar et al., 1994) is the mismatch repair mechanism that corrects spontaneous and postreplicative base alterations and thus is an important pathway for avoidance of muta- tion in normal cells. Genetic defects in mismatch repair mechanisms lead to microsatellite DNA and instability, with subsequent alteration in the stabilization of the genome itself (Modrich, 1994). Enhanced mitogenesis may also trigger more dramatic genetic alterations including mitotic recombination, gene conversion, and nondisjunction. These genetic changes result in further progressive genetic alterations with a high likelihood of resulting in cancer. The types of mutational events, the numbers of such mutations, and the cellular responses to them thus become important factors in our understanding of the mechanisms of chemical carcinogenesis.