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

Summary

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect 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.

Introduction

The alkaline comet assay measures DNA strand breaks at the single-cell level. Suspensions of single cells are embedded in agarose on a microscope slide and the cells lysed to form nucleoids, which contain supercoiled loops of DNA. Electrophoresis at pH>13 results in the loss of supercoiling in DNA loops containing strand breaks, with the freed strands of DNA migrating toward the anode, creating comet-like structures that can be observed by fluorescence microscopy. Fragmented DNA migrates from the "head" of the comet into the "tail" based on the size of the fragment, and the relative fluorescence of the comet tail compared to the total intensity (he....

Protocol

Ethics statement: Procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the US FDA/National Center for Toxicological Research.

NOTE: The study design described here is based on the protocol developed by the Japanese Center for the Validation of Alternative Methods (JaCVAM) for their validation of the in vivo rodent alkaline comet assay¹⁸, and further modified based on recommendations in OECD guideline TG489¹⁹.

1. Preparation

1. Slide preparation
   1. Dissolve regular melting agarose at 1% (w/v) in phosphate buff....

Results

The in vivo alkaline comet assay was performed in conjunction with the enzyme-modified comet assay to measure both direct and oxidative DNA damage in the liver of rats treated with cyproterone acetate (CPA)⁵. CPA is a synthetic hormonal drug that induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females²⁴. We found that the direct DNA damage produced by CPA in the liver of ma.......

Discussion

This protocol describes the concurrent measurement of both direct and oxidative DNA damage in rat liver at the single cell level. The general protocol is applicable to any tissue from which single cells or nuclei can be isolated with minimal processing-induced DNA damage (i.e., DNA damage induced not by the test agent, but by the handling and processing of the animal tissues). In our research, we have conducted alkaline comet assays on cells from bone marrow^7,9, stomach⁶, kidney⁹.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coverslips (No. 1, 24 x 50 mm)	Fisher	12-544-14
Microscope Slides	Fisher	12-550-123
Dimethylsulfoxide (DMSO)	Fisher	67-68-5
EDTA, Disodium	Fisher	BP120-1
Phosphate buffered saline	Fisher	ICN1860454
1x Hanks Balanced Salt Solution (HBSS) (Ca2+, Mg2+ free)	HyClone	SH30588.02
HEPES	Fisher	BP310-1
Low Melting Point Agarose (LMP)	Lonza	50081	NuSieve GTG Agarose
Normal Melting Agarose (NMA)	Fisher	BP1356-100
pH testing paper strips (pH 7.5-14)	Fisher	M95873
Potassium Cloride	Fisher	7447-40-7
Potassium Hydroxide	Fisher	1310-58-3
slide labels (0.94 x 0.5 in.)	Fisher	NC9822036
Sodium Chloride (NaCl)	Fisher	7647-14-5
Sodium Hydroxide (NaOH)	Fisher	1310-73-2
SYBR™ Gold	Invitrogen	S11494
Triton X-100	Fisher	9002-93-1
Trizma Base	Fisher	77-86-1
2.0 ml microcentrifuge tubes	Fisher	05-402-6
Cell strainer (40 µm)	Fisher	22363547
Endonuclease III (Nth)	New England Biolabs	M0268S	Dilution 1:1,000
hOGG1	New England Biolabs	M0241S	Dilution 1:1,000