Benzo [a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC-MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the large amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo [a]pyrene. This exposure caused 0.4-0.8 lesions/ 10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.