Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.