Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Summary

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

Abstract

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

Introduction

The comet assay is increasingly used as a means to evaluate DNA damage in cultured cells and tissues exposed to chemicals or other environmental stressors¹. The assay can detect DNA double- and single-strand breaks, alkali-labile lesions, and single-strand breaks associated with incomplete DNA repair. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline for pharmaceutical testing recommends a DNA strand breakage assay such as the comet assay as a second test to supplement the rodent erythrocyte micronucleus assay for assessing in vivo genotoxicity and as a follow-up test for ass....

Protocol

Tissues were harvested during the conduct of studies performed at AAALAC-accredited facilities at NTP contract laboratories in accordance with Good Laboratory Practice regulations (21 CFR Part 58) and animal use protocols approved by the Institutional Animal Care and Use Committee (IACUC) at each laboratory.

1. Tissue Harvest and Processing

NOTE: It is useful to prepare duplicate sample tubes (e.g., liver) or transfer approximately half of a sample to another storage tube (e.g., duodenum, stomach) to enable reanalysis, if necessary. To minimize potential sample-to-sample variability for intestinal ....

Results

Study 1
Liver was harvested from two cohorts of male Sprague Dawley rats administered corn oil for 4 days, staggered by one week. Slides were prepared from freshly minced tissue, frozen minced tissue, and frozen cubed tissue processed in Merchant’s medium or mincing solution using the tissue mincing device. Frozen tissues obtained from animals from the first cohort were evaluated after freezer storage for ~3.5 months. Frozen tissues obtained from animals from the second cohort were evaluated .......

Discussion

As demonstrated previously^7,12,13, properly handled flash frozen minced tissue provides good results in the comet assay. In fact, baseline % tail DNA values for frozen minced rat and mouse liver prepared in our laboratory are typically ≤6%, as recommended by the OECD test guideline⁹ for freshly minced rat liver samples. Good results have been obtained by multiple laboratories using a variety of tiss.......

Materials

Name	Company	Catalog Number	Comments
Cryovials	Corning Costar	430488
Dental Wax Sheets	Electron Microscopy Sciences	72670
Dissecting (Mincing) Micro Scissors	Fisher Scientific	08-953-1B
DMSO	Sigma-Aldrich	D8418
Hank's Balanced Salt Solution	Gibco	14175-079
KCl	Teknova	P0315-10
KH2PO4	Sigma-Aldrich	P9791
Low Melting Point Agarose	Lonza	50081
Microfuge Tubes (1.7 mL )	Corning	3207
Na2EDTA	Sigma-Aldrich	E5134
Na2HPO4	Sigma-Aldrich	S7907
NaCl	Sigma-Aldrich	S6191
Neutral Buffered Formalin	Leica	600
Scalpel Blades	Miltex	4-110
Syringe Plunger (1 mL )	Fisher Scientific or Vitality Medical	14-826-88; 8881901014	Becton Dickinson or Monoject tuberculin syringe
Tissue Mincing Device	NorGenoTech (Oslo, Norway)	None	Small variability in diameter observed which can affect snuggness of plunger.
Tweezers, plastic	Trade Winds Direct	DF8088N	Reinforced nylon, nonsterile, blunt tip, autoclavable; tradewindsdirect.com
Weigh Boats	Krackler Scientific/Heathrow Scientific	6290-14251B