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 d iethylstilbestrol reactive metabolites by mitochondrial enzymes in vitro. I n vitro, these reactive intermediates bind to mitochondrial DNA. Here we ha ve investigated the in vivo formation of diethylstilbestrol adducts with mi tochondrial 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 hamster s produced several adducts in mitochondrial DNA of both kidney and liver. T he total relative adduct levels were 5- to 6-fold higher in mitochondrial D NA than in nuclear DNA. The chromatographic mobility of mitochondrial DNA a dducts formed in vivo were similar to that of dGMP-DES quinone adducts form ed in vitro. The identity of mitochondrial DNA adducts formed in vivo was f urther confirmed as dGMP-diethylstilbestrol quinone adducts by rechromatogr aphy and cochromatography. Using a DNA polymerase arrest assay we found tha t the DES quinone attack on a mitochondrial respiratory gene, i.e., the gen e 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 t ranscripts was 5- to 10-fold higher in tumor samples than age-matched contr ol kidneys. These findings suggest that i) mitochondrial DNA appears more s usceptible to formation of diethylstilbestrol adducts than nuclear DNA, ii) the DNA adducts formed by DES were predominantly with guanines, iii) the a dducted bases stopped DNA polymerase-mediated ill vitro replication of the COIII gene, and iv) long-term exposure of hamsters to diethylstilbestrol el evated 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 mito chondrial structural abnormality.