Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK(+/-) human lymphoblastoid cells.
Coumestrol, a phytoestrogen found in high levels in alfalfa and red clover, is of concern since endocrine disorders have been observed in farm animals exposed to high levels of phytoestrogens. Previous studies found that coumestrol was an effective inducer of DNA strand breaks, micronuclei, and mutations in the Hypoxanthine phosphoribosyl transferase (HPRT) gene of Chinese hamster ovary cells. In the experiments presented here, we extended the previous studies to examine the effect of coumestrol exposure on AHH-1 TK(+/-) human lymphoblastoid cells. Micronuclei were induced with the highest frequency occurring at day 2 after exposure. Flow cytometric analysis of annexin V-FITC-7-aminoactinomycin D stained cells indicated that the primary pathway of cell death was by apoptosis. Mutations were induced in the Thymidine Kinase (TK) gene and were due primarily to the induction of clones with the slow-growth phenotype. Subsequent molecular analysis revealed the loss of exon 4 in the coumestrol-induced clones, indicative of loss-of heterozygosity and consistent with a proposed inhibition of topoisomerase-II activity as a mechanism of action for coumestrol. Taken together, these results suggest that coumestrol exhibits both mutagenic and clastogenic properties in cultured human lymphoblastoid cells.
Domon
O E
OE
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research (NCTR), US Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
McGarrity
L J
LJ
Bishop
M
M
Yoshioka
M
M
Chen
J J
JJ
Morris
S M
SM
D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D003375:Coumestrol / Q000633:toxicity*; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D006801:Humans; D019656:Loss of Heterozygosity; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015162:Micronucleus Tests; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics
D017931:DNA Primers; D013937:Thymidine Kinase; D003375:Coumestrol