<em>In Vivo</em> Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Abstract

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

The development and validation of EpiComet‐Chip, a modified high‐throughput comet assay for the assessment of DNA methylation status

“…We, and others, have previously described the use of the standard alkaline comet assay in vitro , on several human and nonhuman cell lines, and in vivo using animal models, suggesting that we would not have difficulty performing the comet assay to assess for DNA damage in the immortalized human cells lines that were selected for this study [Collins, ; Azqueta et al, ; Ding et al, ; Weingeist et al, ; Ding et al, ; Ge et al, ; Manjanatha et al, ; Azqueta and Dusinska, ; Ge et al, ; Agnihothram et al, ; Ding et al, ]. However, to formally test this hypothesis, we examined the ability of a known genotoxicant, methyl methanesulfonate (MMS) to induce dose‐dependent induction of DNA damage that we could identify using the standard protocol for the alkaline comet assay.…”

“…After the lysis, the standard procedure was modified to allow for the determination of global DNA methylation status. Briefly, similar to the principles and procedure for the modified oxidative comet assay [Collins and Dusinska, ; Ding et al, ], slides or CometChip preparations were washed in a unique Wash Buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9) and briefly allowed to equilibrate with wash buffer at room temperature. Subsequently, samples were incubated at 37°C in a preheated damp chamber for 105 min by layering either the following: (A) Control treatment buffer (wash buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9) plus 100 μg/mL BSA and 1 mM GTP (required for McrBC enzymatic activity)), or (B) methylation‐specific buffer (control treatment buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9, 100 μg/ml BSA, 1 mM GTP) plus 0.035U/μl McrBC (New England Biolabs, Ipswich, MA, USA) and covering the enzyme solution with cover slips.…”

“…Currently, no single platforms exist that are capable of evaluating, at the cellular level, the genotoxicity (DNA damage), and changes in global DNA methylation status (5 mC%) that occur as a result of exogenous exposures. We, and others, have previously described the use of the standard alkaline comet assay in vitro , on several human and nonhuman cell lines, and in vivo using animal models, as a powerful approach to assess genotoxicity [Collins, ; Azqueta et al, ; Ding et al, ; Weingeist et al, ; Ding et al, ; Ge et al, ; Manjanatha et al, ; Azqueta and Dusinska, ; Ge et al, ; Agnihothram et al, ; Ding et al, ]. The comet assay is amenable to several modifications, including the addition of a subsequent restriction digest step after lysis in the traditional protocol.…”

High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines

“…After DNA was separated in alkaline rinse buffer at 9 V for 15 mins, slides were washed with distilled water twice and stained with 10 μg/ml propidium iodide (Dojindo) at 4°C for 30 min. Fluorescent images were captured using an inverted fluorescence microscope (BZ-9000; KEYENCE, Osaka, Japan), and comet tails of at least 30 cells per slide were analyzed with Open Comet plugin for ImageJ software (NIH, MD, USA) (Ding et al, 2016;Gyori et al, 2014;Olive and Banath, 2006;Rasband, 1997Rasband, -2018Schneider et al, 2012).…”

“…To address the first hypothesis, we evaluated DNA damage by alkaline comet assays (Ding et al, 2016;Olive and Banath, 2006). To compare the effects of doxorubicin on DNA damage between DS-HSA and DR-HSA cells, verapamil was added with doxorubicin to eliminate any discrepancy caused by the drug efflux pump capacities.…”