title: p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells.
abstract: The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.
authors.affiliation: Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA. smorris@nctr.fda.gov
authors.forename: S M
authors.initials: SM
authors.lastname: Morris
authors.forename: J J
authors.initials: JJ
authors.lastname: Chen
authors.forename: O E
authors.initials: OE
authors.lastname: Domon
authors.forename: L J
authors.initials: LJ
authors.lastname: McGarrity
authors.forename: M E
authors.initials: ME
authors.lastname: Bishop
authors.forename: M G
authors.initials: MG
authors.lastname: Manjanatha
authors.forename: D A
authors.initials: DA
authors.lastname: Casciano
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002273:Carcinogens / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects; D005434:Flow Cytometry; D016158:Genes, p53 / Q000235:genetics*; D019833:Genistein / Q000633:toxicity*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D015162:Micronucleus Tests; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D013866:Thioguanine / Q000494:pharmacology; D014271:Trifluridine / Q000494:pharmacology
chemical_list: D002273:Carcinogens; D009153:Mutagens; D019833:Genistein; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D014271:Trifluridine