Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process

Pharmacodynamic Modeling of Anti-Cancer Activity of Tetraiodothyroacetic Acid in a Perfused Cell Culture System

Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations

Electronic cigarette power affects count concentration and particle size distribution of vaping aerosol.

IntroductionElectronic cigarettes (EC) have evolved rapidly toward higher powered devices that produce more vaping aerosol and a more satisfying vaping experience. This research characterized the particle size distribution and estimated the mass concentration of vaping aerosols produced at power outputs spanning the operating range typical of second generation variable voltage EC devices.MethodsEC aerosol was characterized from a single coil atomizer powered by a variable voltage EC battery at the minimum and maximum dial settings (3.3, 11.2 Watts, W), and a lab controlled power supply (3-11.9 W). Aerosol particle size distribution was measured by a Scanning Mobility Particle Sizer and Aerodynamic Particle Sizer, spanning 16 nm to 19.8 μm. A mouth puff was simulated using a 100 mL glass syringe.ResultsConsistent with prior studies, sub-micron EC aerosol size distributions were bimodal, with peaks at 40 and 200 nm, however a previously unreported third mode was observed at approximately 1000 nm. The ~1000 nm mode accounted for 7-20x the aerosol mass of the smaller modes. Increasing atomizer power decreased count concentration of particles 600 nm. Particle mass distribution shifted toward micron sized particles with increasing power and increased the respirable fraction of aerosol, likely due to increased coagulation and condensation around nano-sized particles.ConclusionsVaping power greatly affects EC aerosol count and mass distribution. Mouth puffed EC aerosol spans a much wider particle size range than previously reported, although the major portion of the mass is still well within the alveolar size range the larger particles will deposit within the oro-pharyngeal cavity at 2-3x greater efficiency than in alveoli. These observations have major clinical implications, as aerosol particle size distribution determines deposition sites along the respiratory tract. The results of this experiment stress the need for further research to inform the design, regulation and use of e-cigarette products

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

<div><p>Background</p><p>Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs.</p><p>Objective</p><p>The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells.</p><p>Methods</p><p>Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively.</p><p>Results</p><p>EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage.</p><p>Conclusions</p><p>Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public.</p></div