Detection of DNA double‐strand breaks by pulsed‐field gel electrophoresis

Kawashima, Yuri; Yamaguchi, Nahomi; Teshima, Rie; Narahara, Hisashi; Yamaoka, Yoshio; Anai, Hirofumi; Nishida, Yoshihiro; Hanada, Katsuhiro

doi:10.1111/gtc.12457

Cited by 25 publications

(15 citation statements)

References 34 publications

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“…Briefly, C. jejuni were grown in MH broth alone as well as MH broth supplemented with different concentrations of deoxycholate, after which, pulsed-field gel electrophoresis (PFGE) was performed to detect DNA double strand breaks. The basis of this assay is that the fragmented DNA migrates in the agarose gel during PFGE, whereas the intact DNA does not migrate in the gel 16 . Fragmented DNA was readily visualized in samples prepared from C. jejuni grown in medium containing ≥0.1% deoxycholate (Fig.…”

Section: Resultsmentioning

confidence: 99%

The food-borne pathogen Campylobacter jejuni depends on the AddAB DNA repair system to defend against bile in the intestinal environment

Gourley

Negretti

Konkel

2017

Sci Rep

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Accurate repair of DNA damage is crucial to ensure genome stability and cell survival of all organisms. Bile functions as a defensive barrier against intestinal colonization by pathogenic microbes. Campylobacter jejuni, a leading bacterial cause of foodborne illness, possess strategies to mitigate the toxic components of bile. We recently found that growth of C. jejuni in medium with deoxycholate, a component of bile, caused DNA damage consistent with the exposure to reactive oxygen species. We hypothesized that C. jejuni must repair DNA damage caused by reactive oxygen species to restore chromosomal integrity. Our efforts focused on determining the importance of the putative AddAB DNA repair proteins. A C. jejuni addAB mutant demonstrated enhanced sensitivity to deoxycholate and was impaired in DNA double strand break repair. Complementation of the addAB mutant restored resistance to deoxycholate, as well as function of the DNA double strand break repair system. The importance of these findings translated to the natural host, where the AddAB system was found to be required for efficient C. jejuni colonization of the chicken intestine. This research provides new insight into the molecular mechanism utilized by C. jejuni, and possibly other intestinal pathogens, to survive in the presence of bile.

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Section: Resultsmentioning

confidence: 99%

The food-borne pathogen Campylobacter jejuni depends on the AddAB DNA repair system to defend against bile in the intestinal environment

Gourley

Negretti

Konkel

2017

Sci Rep

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“…Radiomimetic compounds (used in chemotherapy) also cause double-strand breaks and simple complex damage. Agents like bleomycin cause double-strand breaks through oxidized lesions (Regulus et al 2007), while agents like etoposide and cisplatin cause double-strand breaks by interfering with DNA replication forks (Kawashima et al 2017).…”

Section: Generalizability To Other Agentsmentioning

confidence: 99%

Adverse outcome pathways for ionizing radiation and breast cancer involve direct and indirect DNA damage, oxidative stress, inflammation, genomic instability, and interaction with hormonal regulation of the breast

Helm

Rudel

2020

Arch Toxicol

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Knowledge about established breast carcinogens can support improved and modernized toxicological testing methods by identifying key mechanistic events. Ionizing radiation (IR) increases the risk of breast cancer, especially for women and for exposure at younger ages, and evidence overall supports a linear dose-response relationship. We used the Adverse Outcome Pathway (AOP) framework to outline and evaluate the evidence linking ionizing radiation with breast cancer from molecular initiating events to the adverse outcome through intermediate key events, creating a qualitative AOP. We identified key events based on review articles, searched PubMed for recent literature on key events and IR, and identified additional papers using references. We manually curated publications and evaluated data quality. Ionizing radiation directly and indirectly causes DNA damage and increases production of reactive oxygen and nitrogen species (RONS). RONS lead to DNA damage and epigenetic changes leading to mutations and genomic instability (GI). Proliferation amplifies the effects of DNA damage and mutations leading to the AO of breast cancer. Separately, RONS and DNA damage also increase inflammation. Inflammation contributes to direct and indirect effects (effects in cells not directly reached by IR) via positive feedback to RONS and DNA damage, and separately increases proliferation and breast cancer through pro-carcinogenic effects on cells and tissue. For example, gene expression changes alter inflammatory mediators, resulting in improved survival and growth of cancer cells and a more hospitable tissue environment. All of these events overlap at multiple points with events characteristic of "background" induction of breast carcinogenesis, including hormone-responsive proliferation, oxidative activity, and DNA damage. These overlaps make the breast particularly susceptible to ionizing radiation and reinforce that these biological activities are important characteristics of carcinogens. Agents that increase these biological processes should be considered potential breast carcinogens, and predictive methods are needed to identify chemicals that increase these processes. Techniques are available to measure RONS, DNA damage and mutation, cell proliferation, and some inflammatory proteins or processes. Improved assays are needed to measure GI and chronic inflammation, as well as the interaction with hormonally driven development and proliferation. Several methods measure diverse epigenetic changes, but it is not clear which changes are relevant to breast cancer. In addition, most toxicological assays are not conducted in mammary tissue, and so it is a priority to evaluate if results from other tissues are generalizable to breast, or to conduct assays in breast tissue. Developing and applying these assays to identify exposures of concern will facilitate efforts to reduce subsequent breast cancer risk.Biological systems studied This review article summarizes and synthesizes data from cell-culture, experimental animals, and human epidem...

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“…In cases to examine cell cycle effects on DSB formation, 20 mM thymidine was incubated for 12 hr before infection, and then, accumulation of DSBs was analyzed by PFGE after 24‐hr incubation (Figure c). Conditions for PFGE were previously described (Hanada et al., , ; Kawashima et al., ). Briefly, cells were harvested after 6, 12, 24, 48 and 72 hr of incubation with periodontal pathogens and bacterial supernatant and embedded in 0.5% agarose plugs.…”

Section: Methodsmentioning

confidence: 99%

Aggregatibacter actinomycetemcomitans infection causes DNA double‐strand breaks in host cells

et al. 2018

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Periodontal disease, an inflammatory disease, is caused by infection with periodontal pathogens. Long-term periodontal disease increases the risk of oral carcinogenesis. Similar to other peptic cancers, oral carcinogenesis also requires multiple genome instabilities; however, the risk factors related to the accumulation of genome instabilities are poorly understood. Here, we suggested that specific periodontal pathogens may increase the risk of genome instability. Accordingly, we screened several periodontal pathogens based on the ability to induce DNA double-strand breaks (DSBs) in host cells. We found that Aggregatibacter actinomycetemcomitans Y4 infection induced DSB formation in host cells. To assess whether DSB formation induced by infection with A. actinomycetemcomitans occurred through apoptotic chromosome fragmentation, cells were treated with a caspase inhibitor, Z-VAD-FMK. DSB accumulation induced by infection with A. actinomycetemcomitans was observed, even in the presence of Z-VAD-FMK, suggesting that this breakage occurred independently of apoptosis. These results suggested that some periodontal pathogens can increase the risk of genome instabilities in host cells and subsequently increase the risk of carcinogenesis.

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Detection of DNA double‐strand breaks by pulsed‐field gel electrophoresis

Cited by 25 publications

References 34 publications

The food-borne pathogen Campylobacter jejuni depends on the AddAB DNA repair system to defend against bile in the intestinal environment

The food-borne pathogen Campylobacter jejuni depends on the AddAB DNA repair system to defend against bile in the intestinal environment

Adverse outcome pathways for ionizing radiation and breast cancer involve direct and indirect DNA damage, oxidative stress, inflammation, genomic instability, and interaction with hormonal regulation of the breast

Aggregatibacter actinomycetemcomitans infection causes DNA double‐strand breaks in host cells

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