The γH2AX Assay for Genotoxic and Nongenotoxic Agents: Comparison of H2AX Phosphorylation with Cell Death Response

Nikolova, Teodora; Dvorak, Mirek; Jung, Fabian; Adam, Isabell; Krämer, Elisabeth; Gerhold‐Ay, Aslihan; Kaina, Bernd

doi:10.1093/toxsci/kfu066

Cited by 109 publications

(85 citation statements)

References 58 publications

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“…Phosphorylation of histone H2AX at serine 139 (producing γ‐H2AX) is a well‐established biomarker of DNA damages, particularly DNA double‐strand breaks (Pilch et al, ; Rogakou, Pilch, Orr, Ivanova, & Bonner, ). γ‐H2AX has been used in multiple scientific fields, e.g., in vitro assessment of preclinical drugs and genotoxicity screening for chemical materials (Khoury, Zalko, & Audebert, ; Nikolova et al, ; Watters, Smart, Harvey, & Austin, ), and is rapidly accumulated over a large region of chromatin surrounding double‐strand breaks (Rogakou, Boon, Redon, & Bonner, ), leading to aggregation of repair proteins; this process can be microscopically detected by immunofluorescence and immunohistochemistry using specific primary antibodies (Redon et al, ). Therefore, immunostaining of γ‐H2AX is expected to be applied as a useful tool to predict in vivo genotoxicity of chemicals (Nagelkerke & Span, ; Thompson et al, ; Toyoda et al, ).…”

Section: Discussionmentioning

confidence: 99%

γ‐H2AX formation in the urinary bladder of rats treated with two norharman derivatives obtained from o‐toluidine and aniline

Toyoda

Totsuka²,

Matsushita

et al. 2017

J of Applied Toxicology

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Aminomethylphenylnorharman (AMPNH) and aminophenylnorharman (APNH) are mutagenic norharman derivatives obtained from o-toluidine and aniline, respectively. APNH is carcinogenic to the urinary bladder of rats and present in urine samples of healthy volunteers, indicating that norharman derivatives may be associated with cancer development in the urinary bladder of humans. To evaluate the possible role of AMPNH and APNH in bladder carcinogenesis, we examined the formation of γ-H2AX, a DNA damage response marker, in the urinary bladder of rats. Seven-week-old male F344 rats were treated with 400 ppm AMPNH or 40 ppm APNH in the diet for 4 weeks. Animals were killed at the end of administration or after 2 weeks of recovery, and immunohistochemistry for γ-H2AX and Ki67, a cell proliferation marker, was performed. At week 4, γ-H2AX formation in bladder epithelial cells was significantly increased by APNH treatment as compared with that in controls. AMPNH also induced upregulation of γ-H2AX formation, although there was no statistical significance. After the recovery period, γ-H2AX-positive cells were reduced but remained significantly higher in AMPNH and APNH groups than in the control group. Ki67-positive cells were significantly increased by AMPNH and APNH at week 4 and reduced to the same level as the control after 2 weeks of recovery. Expression of KRT14, a bladder stem cell marker, was also increased in the basal layer by the two norharman derivatives. Thus, AMPNH and APNH showed in vivo genotoxicity in the bladder epithelium of rats, and APNH may be a potent causative agent of bladder carcinogenesis.

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Section: Discussionmentioning

confidence: 99%

γ‐H2AX formation in the urinary bladder of rats treated with two norharman derivatives obtained from o‐toluidine and aniline

Toyoda

Totsuka²,

Matsushita

et al. 2017

J of Applied Toxicology

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“…The proteins, such as c-H2AX, ATM, CDKN1A, and TP53, involved in the early steps of the cellular response in sensing DNA damage and controlling progression, have been proposed as the top group of candidate biomarkers (Marchetti et al, 2006). Due to the signal amplification of c-H2AX foci at the site of DNA damage, the detection of c-H2AX has been identified as an early sensitive cellular biomarker of DNA damage (Nikolova et al, 2014;Rogakou et al, 1998). In this study, we developed a multi-parametric HCA assay along with the detection of c-H2AX and found that BPA and its analogues significantly altered the expression levels of c-H2AX.…”

Section: Discussionmentioning

confidence: 99%

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

Liang

Yin

et al. 2016

Toxicol. Sci.

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Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose-and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.

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“…There is usually a good correlation between drug doses and γ H2AX formation in vitro 23, 24. However, we observed different γ H2AX/apoptosis kinetics between fibroblasts, keratinocytes, and melanoma in the MSM that suggests a differential DNA damage response that is cell‐type dependent.…”

Section: Discussionmentioning

confidence: 63%

Evaluation of surrogate tissues as indicators of drug activity in a melanoma skin model

et al. 2016

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The development of novel cancer treatments is a challenging task, partly because results from model systems often fail to predict drug efficacy in humans, and also tumors are often inaccessible for biochemical analysis, preventing effective monitoring of drug activity in vivo. Utilizing a model system, we evaluated the use of drug‐induced DNA damage in surrogate tissues as indicators of drug efficacy. Samples of a commercially available melanoma skin model (Mattek MLNM‐FT‐A375) containing keratinocyte and fibroblast layers with melanoma nodules were subjected to various chemotherapeutic regimens for one, four, or eight days. At these times they were analyzed for DNA double‐stranded breaks (γH2AX foci) and apoptosis (TUNEL). A wide range of drug responses in both tumor and normal tissues were observed and cataloged. For the melanoma, the most common drug response was apoptosis. The basal keratinocyte layer, which was the most reliable indicator of drug response in the melanoma skin model, responded with γH2AX foci formation that was abrupt and transient. The relationships between tumor and surrogate tissue drug responses are complex, indicating that while surrogate tissue drug responses may be useful clinical tools, careful control of variables such as the timing of sampling may be important in interpreting the results.

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The γH2AX Assay for Genotoxic and Nongenotoxic Agents: Comparison of H2AX Phosphorylation with Cell Death Response

Cited by 109 publications

References 58 publications

γ‐H2AX formation in the urinary bladder of rats treated with two norharman derivatives obtained from o‐toluidine and aniline

γ‐H2AX formation in the urinary bladder of rats treated with two norharman derivatives obtained from o‐toluidine and aniline

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

Evaluation of surrogate tissues as indicators of drug activity in a melanoma skin model

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