Site Specific Mutagenesis by DNA Adducts

Exposure of cells to DNA damaging agents usually results in the formation of a vast population of structurally heterogeneous carcinogen-DNA adducts. It is hypothesized that misreplication of adducts initiates cells along the pathway to malignancy. A large body of evidence indicates, however, that only a subset of the adduct population is likely to contribute to mutagenicity and carcinogenicity. When we began our work, a central problem in the field of carcinogenesis was the lack of an experimental system to identify which DNA lesion gave rise to which types of mutations. The laboratory established such a system.

The genome of a virus or plasmid is processed by using recombinant DNA techniques to situate a small gap at a specific site. An oligonucleotide containing a single DNA adduct is then synthesized and ligated into the gap. The site specifically modified genome is introduced into a bacterial or mammalian cell, allowed to replicate intra- or extra-chromosomally and, finally, progeny are isolated. Reduction in the yield of progeny is an indication of the genotoxicity of the adduct. We also determine the type, amount, and genetic requirements for mutagenesis induced by the adduct. This technology enables one to rank the mutagenic and genotoxic potentials of the various adducts that form in the genomes of cells treated with DNA damaging agents. Using this system we have defined the genetic effects of the DNA lesions induced by oxidants and ionizing radiation, simple alkylating agents, aflatoxin B1, cisplatin, 4-aminobiphenyl, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and vinyl chloride.

Future studies will be in three areas: (i) understanding how aflatoxin B1 (AFB1) causes mutations that give rise to tumors, (ii) understanding how oxidized DNA bases give rise to mutations and (iii) trying to apply knowledge of mutagenesis toward the development of safer drugs.

AFB1 is a major cause of concern worldwide owing to its ability to induce liver cancer. In earlier studies we showed that the exo-epoxide of AFB1 gives rise to its DNA adducts, and recently we showed that the N7 guanine adduct formed by the epoxide has a mutational signature that matches (in terms of genetic requirements) that of the toxin in cellular systems. Human tumors from Asia and Africa, where exposure to AFB1 is a major problem, show the same mutation as seen in our studies. In the epidemiological studies, p53 genes from tumors showed a G to T transversion and, interestingly, most of those studies show a striking clustering of the transversions at a specific position in the protein -- at codon 249. We plan to study the mechanism by which this hotspot arises. Specifically, we are constructing shuttle vectors that will contain the individual AFB1-DNA adducts at each site of possible adduct formation in the region of the p53 mutational hotspot. The extreme lability of N7 guanyl adducts has prevented the study of many of AFB1 adducts in this or any sequence context. Fortunately we were able to overcome that obstacle due to a recent chemical advance, and we are now able to synthesize the desired oligonucleotides for genome construction. We shall introduce the vectors into human cells and determine whether the mutational context specificity seen in intact organs is also seen in cell culture systems in which the vector integrates into the host genome. One special feature of our work plan is the use of a hepatitis virus driven system in which the promoting effect of viral infection on mutation or selection of mutant cells will be examined; hepatitis B infection is almost always associated with a high risk to aflatoxin carcinogenesis in humans.

Other studies will focus on continuing our study of the mutagenic effects of alkyl and oxidized DNA bases. We identified 8-oxoguanine some years ago as a premutagenic lesion and it is now believed to be the second most frequent contributor to spontaneous mutations in aerobic organisms. Despite much effort the major contributor to spontaneous adduct driven mutations has evaded identification. We are involved in a systematic search for novel mutagenic lesions formed by oxidation.

By identifying the DNA adducts of environmental carcinogens and drugs that are deleterious to humans, we can implement intervention strategies that minimize risk to humans who are exposed to these agents. In the preceding paragraph we indicate how this goal could be achieved with drugs. Using oxidized DNA bases as a second example, knowledge of the lesions giving rise to cancer allows one to identify risk factors (i.e., genetic defects in the repair of that adduct) and even strategies to suppress the formation of the genotoxic adducts.