| Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues. | |
| | |
MedLine Citation:
|
PMID: 11999389 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 units/g liver at 37 degrees C for 20 min) for isolation of intact cells for subsequent comet assay We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation (-85 degrees C for 72 h). To minimize this damage, minced liver (1.0 g) and kidney (0.5 g) tissues were added to 20 ml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO (-85 degrees C for 72 h and 3 weeks), and to a slightly lesser extent with glycerol (72 h), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150units/g liver and 300units/g kidney for 10 min produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at - 85 degrees C for 72 h. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay. |
| | |
Authors:
|
Miao-Lin Hu; Cheng-Hung Chuang; Hok-Man Sio; Shu-Lan Yeh |
Related Documents
:
|
5353529 - Quantitative determination of deoxyribonucleic acid in rat brain. 2397479 - Relationship between hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine forma... 6694979 - Liver nuclear dna synthesis in mice following carbon tetrachloride administration or pa... 884109 - Normal endonuclease activities for damaged dna during hepatocarcinogenesis. 10580689 - Accumulation of dna damage in pre- and posthepatectomized liver of aged rats. 23219889 - Genetic transformation of grapevine cells using the minimal cassette technology: the ne... 3058329 - High frequency flp-independent homologous dna recombination of 2 mu plasmid in the yeas... 12605379 - Biomonitoring with the comet assay of greek greenhouse workers exposed to pesticides. 16936559 - Lack of decay of hiv-1 in gut-associated lymphoid tissue reservoirs in maximally suppre... |
Publication Detail:
|
Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
|
Title: Free radical research Volume: 36 ISSN: 1071-5762 ISO Abbreviation: Free Radic. Res. Publication Date: 2002 Feb |
Date Detail:
|
Created Date: 2002-05-09 Completed Date: 2002-10-03 Revised Date: 2006-11-15 |
Medline Journal Info:
|
Nlm Unique ID: 9423872 Medline TA: Free Radic Res Country: Switzerland |
Other Details:
|
Languages: eng Pagination: 203-9 Citation Subset: IM |
Affiliation:
|
Department of Food Science, National Chung-Hsing University, Taichung, Taiwan. mlhuhu@dragon.nchu.edu.tw |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Animals Collagenases / metabolism Comet Assay / methods* Cryopreservation / methods* DNA / chemistry, drug effects, metabolism DNA Damage* / drug effects Dimethyl Sulfoxide / pharmacology Ferric Compounds / pharmacology Freezing Glycerol / pharmacology Kidney / drug effects, metabolism* Liver / drug effects, metabolism* Nitrilotriacetic Acid / analogs & derivatives*, pharmacology Oxidative Stress / drug effects Rats Rats, Sprague-Dawley |
| Chemical | |
Reg. No./Substance:
|
0/Ferric Compounds; 139-13-9/Nitrilotriacetic Acid; 16448-54-7/ferric nitrilotriacetate; 56-81-5/Glycerol; 67-68-5/Dimethyl Sulfoxide; 9007-49-2/DNA; EC 3.4.24.-/Collagenases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt...
Next Document: Scavenging of benzylperoxyl radicals by carotenoids.