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p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells
S M Morris1, J J Chen, O E Domon
1Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA. smorris@nctr.fda.gov
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.
Insights
Genistein, a soy phytoestrogen, acts as a chromosomal mutagen, inducing DNA damage. The tumor suppressor p53 influences the recovery of these genotoxic effects, potentially by initiating apoptosis.
Area of Science:
- Molecular Biology
- Genotoxicity Studies
- Cancer Research
Background:
- Genistein, a soy isoflavone, is a known inhibitor of tyrosine kinases and topoisomerase-II (topo-II).
- Genistein exposure induces DNA damage in mammalian cells, similar to known topo-II inhibitors.
- The role of the tumor suppressor gene p53 in genotoxic response to genistein is not fully understood.
Purpose of the Study:
- To investigate the genotoxicity of genistein in human lymphoblastoid cells with varying p53 functional status.
- To assess genistein's impact on DNA damage, mutation induction, and cell death pathways.
- To determine if p53 status influences the cellular response to genistein-induced genotoxicity.
Main Methods:
- Exposure of human lymphoblastoid cell lines (AHH-1 tk+/- with p53+/- and L3 with p53+/+) to genistein.
- Quantification of micronuclei induction via microscopic analysis.
- Measurement of mutant fractions at the thymidine kinase (tk) and hypoxanthine phosphoribosyl transferase (hprt) loci.
- Flow cytometry to analyze cell viability, apoptosis, and degeneration rates.
Main Results:
- Genistein induced micronuclei in both cell lines, indicating chromosomal damage.
- A significant increase in 6-thioguanine (6-TG)-resistant clones (hprt locus) was observed in both cell lines.
- Genistein induced trifluorothymidine (TFT)-resistant clones (tk locus) with slow-growth phenotype only in the p53-mutant AHH-1 cells.
- Cell death occurred primarily via apoptosis, with altered kinetics of cell viability reduction based on p53 status.
Conclusions:
- Genistein exhibits chromosomal mutagenicity.
- The functional status of p53 influences the recovery of chromosomal mutants, potentially through apoptosis signaling.
- Genistein's genotoxic effects are modulated by cellular p53 status.