The objective of this research project is to investigate the possibility that folate deficiency, which is a common human nutritional state, may play an important role in carcinogenesis by promoting genetic instability. Folic acid deficiency is associated with numerous chromosomal changes and enhances the genetic damage caused by mutagens. Therefore, folic acid supplementation may be a cancer control strategy relevant to large populations. The First Specific Aim of this proposal is to determine whether any site or type of DNA lesion predominates in folate deficient cells or the same cells treated with mutagens in vitro. Chinese hamster ovary (CHO) cells will be grown in folate replete or deficient medium; some cells from each nutritional state will be treated with N-ethyl-N- nitrosurea (ENU) or ethyl methanesulfonate (EMS). Molecular analysis of the mutations will be performed by extraction of RNA, reverse transcription-PCR, and DNA sequencing. An analysis of the spectrum of mutations associated with folate deficiency may help define the biochemical mechanisms responsible for the genetic damage that causes human cancer. Furthermore, the mutational spectrum may be useful in molecular epidemiologic studies to assess risk in populations. The Second Specific Aim is to measure the effect of folate deficiency on genetic damage in vivo (including mutational spectra) in rats following administration of chemotherapeutic drugs. Weanling Fischer 344 rats will be divided into 3 dietary groups---marked folate deficiency, moderate folate deficiency and folate replete---and treated with either ENU or Cytoxan. Frequency of somatic mutations at the hprt locus will be determined using a clonal assay. Molecular analyses, will be performed on mutant clones. Mutant frequency and molecular spectra differences will be determined between the groups. The goals of this Aim are to provide evidence that a close relationship exists between folate status and chemotherapy-induced genetic damage and to define the effect of folate deficiency on mutational spectra in vivo. The Third Specific Aim is to determine if folate nutritional status (deficient, sufficient or supplemented) modulates the efficacy or toxicity of chemotherapeutic drugs in vivo. Rats in the 3 dietary groups will be injected with a rat mammary tumor and treated with Cytoxan, 5-FU or Adriamycin. Tumor growth inhibition will be measured and compared between the groups to determine if folate status affects the growth of the tumor in vivo or alters the efficacy of the anti-tumor drugs. Other rats will be treated with LD50 drug doses and observed for signs of toxicity. Support of the hypothesis by the results of these experiments would indicate that folic acid ameliorates genetic damage caused by mutagens and is a safe and effective candidate drug for chemoprevention trials in humans.
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