Base sequence-specific attack of stilbene estrogen metabolite(s) on the mitochondrial DNA: Implications in the induction of instability in the mitochondrial genome in the kidney of Syrian hamsters

We have demonstrated previously that diethylstilbestrol is metabolized to diethylstilbestrol reactive metabolites by mitochondrial enzymes in vitro. In vitro, these reactive intermediates bind to mitochondrial DNA. Here we have investigated the in vivo formation of diethylstilbestrol adducts with mitochondrial DNA, the nature of mitochondrial DNA-diethylstilbestrol adducts, and the influence of diethylstilbestrol adduction on in vitro replication of a mitochondrial gene. Diethylstilbestrol administration to male hamsters produced several adducts in mitochondrial DNA of both kidney and liver. The total relative adduct levels were 5- to 6-fold higher in mitochondrial DNA than in nuclear DNA. The chromatographic mobility of mitochondrial DNA adducts formed in vivo were similar to that of dGMP-DES quinone adducts formed in vitro. The identity of mitochondrial DNA adducts formed in vivo was further confirmed as dGMP-diethylstilbestrol quinone adducts by rechromatography and cochromatography. Using a DNA polymerase arrest assay we found that the DES quinone attack on a mitochondrial respiratory gene, i.e., the gene for subunit III of cytochrome c oxidase (COIII), was specific for guanine residues that were adjacent to cytosine residues. Long-term treatment with diethylstilbestrol produced tumors in the kidney, and the level of COIII transcripts was 5- to 10-fold higher in tumor samples than age-matched control kidneys. These findings suggest that i) mitochondrial DNA appears more susceptible to formation of diethylstilbestrol adducts than nuclear DNA, ii) the DNA adducts formed by DES were predominantly with guanines, iii) the adducted bases stopped DNA polymerase-mediated in vitro replication of the COIII gene, and iv) long-term exposure of hamsters to diethylstilbestrol elevated the expression of COIII mRNA. These results suggest that obstruction of replication of the mitochondrial genes by covalent modifications of the mitochondrial DNA by diethylstilbestrol may produce mitochondrial genomic instability in vivo and may provide an explanation for the DES-induced mitochondrial structural abnormality.