Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/ urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPAfiltered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.DNA adducts ͉ DNA strand breaks ͉ tandem repeat mutation C ombustion of fossil fuels results in the production of complex mixtures of chemicals that are released into the environment and potentially affect millions of people globally. Previous work demonstrated that the offspring of wild birds breeding near integrated steel mills on the North American Great Lakes inherited increased numbers of tandem repeat DNA sequence mutations compared with those from areas without steel mills (1, 2). Subsequent studies investigated expanded simple tandem repeat (ESTR) mutation in outbred laboratory mice caged near two integrated steel mills and a major highway in Hamilton, Ontario, Canada, and at a rural reference site (3, 4). Using a pedigree approach (5), a significant increase in germ-line mutation rate was found in mice housed in the industrial environment compared with the reference site. The majority of mutations were transmitted through the paternal germ line. High-efficiency particulate-air (HEPA) filtration of the ambient air resulted in a significant reduction in mutation frequency, down to levels measured at the reference location (4). Therefore, the particulate fraction of air in this industrial location was largely responsible for the mutagenic hazard.These findings show that chemical pollutants may cause heritable mutation. Further research is required to confirm these results...

Urban and industrial air pollution can cause elevated heritable mutation rates in birds and rodents. The relative importance of airborne particulate matter versus gas-phase substances in causing these genetic effects under ambient conditions has been unclear. Here we show that high-efficiency particulate-air (HEPA) filtration of ambient air significantly reduced heritable mutation rates at repetitive DNA loci in laboratory mice housed outdoors near a major highway and two integrated steel mills. These findings implicate exposure to airborne particulate matter as a principal factor contributing to elevated mutation rates in sentinel mice and add to accumulating evidence that air pollution may pose genetic risks to humans and wildlife.

Hundreds of thousands of people worldwide live or work in close proximity to steel mills. Integrated steel production generates chemical pollution containing compounds that can induce genetic damage (1, 2). Previous investigations of herring gulls in the Great Lakes demonstrated elevated DNA mutation rates near steel mills (3, 4) but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden. To address these issues experimentally, we exposed laboratory mice in situ to ambient air in a polluted industrial area near steel mills. Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated steel mills was 1.5-to 2.0-fold elevated compared with those at a reference site 30 km away. This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germline. Our results indicate that human and wildlife populations in proximity to integrated steel mills may be at risk of developing germline mutations more frequently because of the inhalation of airborne chemical mutagens. Integrated steel mills produce chemical mutagens that contaminate atmospheric and aquatic environments (1, 2), and may pose a genetic hazard to humans and wildlife. Herring gulls (Larus argentatus) nesting near steel mills on the Great Lakes were shown to have higher germline mutation rates at minisatellite DNA loci than those at rural sites (3), and mutation frequency increased with colony proximity to integrated steel mills (4). It was postulated that inhaled airborne contaminants emitted from steel mills, such as polycyclic aromatic compounds, were largely responsible for mutation induction; however, contaminants in the aquatic food web and differences in disease and nutritional status among gull colonies could not be eliminated as contributing factors. Therefore, the role of air pollution in producing germline mutations and the risk to humans living near steel mills could not be determined.Rodent expanded simple tandem repeat (ESTR) DNA consists of 4-to 6-bp repeat units in long tandem arrays that are unstable in the germline and tend to mutate by insertion or deletion of a number of repeat units (5-7). Laboratory studies have demonstrated that murine ESTR loci are susceptible to germline mutations induced by chemical (8) or radioactive (9, 10) mutagens, and therefore may be useful tools for environmental contamination studies. The use of sentinel laboratory animals exposed in situ is a powerful experimental approach for assessing air pollution hazards because it combines the controlled elements of laboratory studies with direct exposure to ambient pollution levels (11,12). Here, we compare germline ESTR mutation rates in laboratory mice exposed to ambient air at an industrial site near integrated steel mills to those exposed at a rural reference location, with the objective of testing inhalation of industrial air pollution as a route ...

Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.