Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells

Abstract

Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT). Equilin and equilenin are the major components of the widely prescribed drug used for ERT. These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages. To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF , was exposed to 4-OHEQ and transfected into human fibroblast. Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing. The proportion of plasmids with the mutated supF gene was increased dose-dependently. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions. Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions. The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site. C:G → G:C and C:G → A:T mutations were detected preferentially with lesser numbers of C:G → T:A transitions. Sixty-two percent of base substitutions were observed particularly at C:G pairs in ^5′ -TC/AG- ^5′ sequences. Using ³² P-post-labeling/gel electrophoresis analysis, 4-OHEN–dC was a major adduct, followed by lesser amounts of 4-OHEN–dA adduct. Mutations observed at C:G pairs may result from 4-OHEN–dC adduct. These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in ^5′ -TC/AG- ^5′ sequences.