PERSISTENCE OF DNA ADDUCTS AND MUTAGENESIS

24 May

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-

plasms; however, a statistically significant increased level of ethylation of this base was noted in cancer patients as compared with controls. In a more recent study by Hsieh and Hsieh (1993), DNA adducts of aflatoxin B1 were demonstrated in samples of human placenta and cord blood from patients in Taiwan, an area of high liver cancer incidence (Chapter 11). In addition to the determination of specific structural DNA adducts, the use of the 32P-postlabeling assay to deter- mine the presence of DNA adducts in human tissues has also been exploited (Beach and Gupta,

1992). As expected, a variety of adducts are found in both normal individuals and those poten- tially exposed to specific carcinogenic agents. In addition to DNA adducts, adducts of specific carcinogens  with serum proteins have been demonstrated.  Bryant et al. (1987) have shown a five- to sixfold greater level of hemoglobin adducts of 4-aminobiphenyl  in smokers compared with nonsmokers. While this adduct has a finite lifetime, the chronic exposure to cigarette smoke dramatically increases the level of the adduct, suggesting the use of such determinations in esti- mating exposure to carcinogenic agents.

Thus, the persistence of macromolecular adducts of the ultimate forms of chemical carcin- ogens may be very important in the carcinogenic mechanism of such agents. However, as noted above, such persistence is only one factor in the complex process of cancer development.

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