This project will determine if specific mutations in human subjects can be used as a biomonitor of aflatoxin B1 (AFB1) exposure. The X-linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus will serve as the genetic target in these studies. We will first determine the frequency, molecular nature, and location of hprt mutations in human cells treated with AFB1 in culture. Individual mutant clones will be used to generate hprt cDNA. The cDNA will then be PCR-amplified and sequenced to reveal the AFB1-induced mutations. We will also isolate mutant hprt T- cells from AFB1-treated rats and determine the frequency molecular nature, and location of mutations in hprt exon 3, using established techniques. Exon 3 contains the largest contiguous portion of the hprt coding frame and this exon contains mutational hotspots for a variety of mutagens. Based on the results obtained in vitro and in the rat, we will then develop molecular techniques to specifically detect the most frequent mutations induced by AFB1. These techniques will be PCR-based and will have the sensitivity to detect the presence of specific AFB1 mutations in pooled mutant populations containing up to 100 different hprt mutants. Mutants isolated from individual rats will be pooled and will be probed for the existence of the mutational hotspots observed in tissue culture. This will determine whether mutations induced by AFB1 in vitro can be used to predict in vivo AFB1-induced mutations in the rat. Finally, we will isolate mutant hprt T-cells from human subjects from Quidong Province, People's Republic of China who are exposed to high levels of dietary AFB1. Mutants from each individual will be pooled and probed for the specific AFB1 mutations observed in human cells in culture and in the rat. Frequency results will be compared to low-exposure individuals from the same region. Mutation results obtained in exposed individuals will be correlated with (i) the aflatoxin levels in food, (ii) the level of N7-G-AFB1 adduct in urine, (iii) the quantity of different aflatoxin metabolites in urine, (iv) the levels of aflatoxin-albumin adducts in blood, (v) lymphocytic expression of glutathione-S-transferase mu, and (vi) metabolic phenotypes for cytochrome P450 1A2 and 3A4. These results will test (i) whether in vitro mutational results can be used to predict results obtained in vivo in humans and rodents, (ii) whether mutational spectra can be used to detect human exposure to a potent environmental mutagen, and (iii) whether the metabolic capability of an individual can be correlated with genetic risk.
| Wang, S S; O'Neill, J P; Qian, G S et al. (1999) Elevated HPRT mutation frequencies in aflatoxin-exposed residents of daxin, Qidong county, People's Republic of China. Carcinogenesis 20:2181-4 |
| Cariello, N F; Cui, L; Skopek, T R (1994) In vitro mutational spectrum of aflatoxin B1 in the human hypoxanthine guanine phosphoribosyltransferase gene. Cancer Res 54:4436-41 |
