Journal of Cancer Prevention 2013; 18(4): 277-288
Published online December 31, 2013
https://doi.org/10.15430/JCP.2013.18.4.277
© Korean Society of Cancer Prevention
Seung Hun Kang1,*, Jee Young Kwon1,*, Jong Kwon Lee2, and Young Rok Seo1
1Department of Life Science, Institute of Environmental Medicine for Green Chemistry, Dongguk University, Seoul, 2Toxicological Research Division, National Institute of Food and Drug Safety Evaluation (NIFDS), Korea Food and Drug Administration (KFDA), Cheongwon, Korea
Correspondence to :
Young Rok Seo, Department of Life Science, Dongguk University, 30 Pildong-ro 1-gil, Jung-gu, Seoul 100-715, Korea Tel: +82-2-2260-3321, Fax: +82-2-2290-1392, E-mail: seoyr@dongguk.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genotoxic events have been known as crucial step in the initiation of cancer. To assess the risk of cancer, genotoxicity assays, including comet, micronucleus (MN), chromosomal aberration, bacterial reverse, and sister chromatid exchange assay, can be performed. Compared with
Keywords:
Cancer is the leading cause of human mortality all over the world.1 Most cancer tissues show a number of complex chromosomal aberrations.2,3 The induction and accumulation of genetic damage can cause genomic instability, and it is known as a crucial step in the generation of cancer.4 Oncogenicity studies of carcinogenic potential using genotoxicity assays are on the rise. Altered gene expression, abnormal cell growth, and disruption of normal cell function may be related with the genotoxic effects of industrial carcinogens or other potential genotoxic agents. These phenomenon can result in the genomic instability and possibly carcinogenesis.1 For evaluating risk of cancer, genetic damage can be determined by genotoxicity assays, including comet assay, micronucleus assay, chromosome aberration assay, gamma-H2AX, and bacterial reverse testing. In this review, the micronucleus assay and the comet assay are focused.5 Since comet assay takes advantages of speediness, high sensitivity and flexibility for measuring capacity of DNA-strand breakage at the level of individual cells, and micronucleus assay exhibits highly reliable, rapid, and broad-spectrum determination of DNA damage at chromosome level (e.g. screening of chromosomal instability, DNA repair capacity, nuclear division rate, mitogenic response and incidence of necrotic and apoptotic cells).6
The genotoxicity tests officially approved as the Organization for Economic Co-operation and Development (OECD) test guidelines include the bacterial reverse mutation test, chromosome aberration test, micronucleus test, and sister chromatid exchange assay.
The comet assay firstly established by ?stling and Johanson has been widely applied for studying DNA strand breaks at the single cell level.9 The
Beside comet assay, the MN assay has been developed for genotoxicity and mutagenicity detection testing of chemicals that induce the formation of small membrane bound DNA fragments in cells (well-known as micronucleus).14?16 In principle, the MN assay is capable of detecting potential genotoxic chemicals that can modify chromosome structure and induce segregation error.17 In number of researches, standard laboratory strains of animals used in the MN assay are F344 rat, SD rat, and CD-1 mouse. Recent studies using the MN assay have shown that increased MN frequency is related with cancer risk, thus supporting the evidence that MN can be a biomarker of carcinogenesis.18,19 As so far application of genotoxicity testing suggests that no single assay can fully detect all genotoxic aspects,20 the ICH guidance on genotoxicity testing have thus proposed combining the
Measurement of DNA damage using genotoxicity assays has been known as a crucial approach for understanding the carcinogenesis and assessing the risk of cancer incidence.22,23 This review will discuss the significance of
Any type of animal tissue (e.g. liver, stomach, and blood cells) can be applied to the
Usually a portion of the left lateral lobe of the liver tissue is removed from a whole liver and then washed sufficiently with an ice-cold appropriate mincing buffer. The washed portion is minced to obtain the single cell suspension. The cell suspension is placed on ice to allow the cluster to settle down. Then, the supernatant can be used to make a comet slide. After preparing the comet slide, the slide is incubated with cold alkaline lysis solution overnight. After the lysis process, the slide is rinsed with deionized water to remove residual detergent and salts. After DNA unwinding using an electrophoresis solution, the slide is electrophoresed. After electrophoresis, the slide is neutralized. Then, the slide is dehydrated by absolute ethanol, air dried at room temperature. The slide is stained with SYBR Gold, and the comet can be visualized under a fluorescence microscope and quantitated via image analyzer system such as Comet IV software (Perspectives, UK). For each sample, one hundred comet cells are subjected for quantitative analysis. Excessively damaged comets showing an appearance of “hedgehog” consisting of very small comet heads and largely-diffused comet tails represent dead cells and should not be analyzed. The parameters used in the comet analysis are as follows: % tail DNA, tail length (a distance between the center of the head mass and the center of the tail mass), and olive tail moment [= tail length × DNA in tail (% tail DNA/100)].31
The whole stomach tissue of a sacrificed animal is initially cut opened and washed until free of food residue using an ice-cold mincing buffer. A portion of the forestomach is removed and then discarded, and the glandular stomach is incubated with the ice-cold mincing buffer. After incubation, the surface epithelial layer is gently scraped. The removed layer is discarded, and the mucosa is rinsed with the ice-cold mincing buffer. Then, the epithelia of the glandular stomach are scraped to obtain cell suspension. The cell suspension is placed on ice to allow cluster to settle down. Then, the cell suspension of the supernatant can be used to make the comet slides. The cell suspension and low-melting-agarose gel are mixed, and then the cell/agar mixture is dispensed onto the comet slide.31 As before, the comet slides follow the process of lysis, DNA unwinding, electrophoresis, neutralization, dehydration, DNA staining, and image analysis.
Blood sample can be collected by venipuncture. All blood samples must be immediately cooled and processed within 2 hours after collection. The layer made by the blood sample mixed with low-melting-point agarose is placed on the comet slide. As mentioned above, the comet assay is composed of the process of lysis, DNA unwinding, electrophoresis, neutralization, dehydration, staining of comet slide, and image analysis.32
The micronucleus (MN) can be generally formed by lagging acentric chromosome fragments, acentric chromatid fragments, or whole chromosome that could not included in the daughter nucleus during mitosis telophase because the whole chromosome did not combine with the spindle during the segregation process of anaphase.15,17,33,34 These chromosome fragments surrounded by nuclear membrane are well known as MN, which is morphologically similar to normal nucleus but smaller in size.35 Compared to other genotoxicity assays, the MN assay is a quick and easy assay at the data analysis step. Moreover, it has no requirement for metaphase cells, and shows identifiable cells under a fluorescence microscope because each cell has only one nuclear division.1,36 The
The
The erythrocyte of a blood sample can be collected by bleeding from a blood vessel (e.g. mouse tail vein), cardiac puncture, or large vessel at animal sacrifice. The blood smears are prepared on glass slides and then subsequently stained for microscopy analysis. Using flow cytometry-based analysis, the sample slides should be fixed and stained. DNA-specific staining using acridine orange can exclude the possibility of any artifacts generated by non-DNA-specific staining.38
After liver tissues are removed, it is recommended to immediately perfuse the liver tissues with cold saline solution until the blood is completely removed. The final washing uses a cold homogenizing buffer containing EDTA, NaCl, and DMSO. After weighing the liver sample, the tissues are minced, suspended in cold homogenization buffer, and homogenized maintaining in the cold buffer on ice using a potter-type homogenizator. After centrifugation of the homogenate, the supernatant is removed, and the pellet is resuspended in the homogenization buffer.39 The resuspended pellet needs to be settled down. Then, a drop of the suspension is placed at the end of a pre-cleaned, grease-free, microscopic slide. Subsequently, the drop is spread into a single cell layer without damaging the cell morphology, using a clean cover glass held at 45 degrees.39,40 Next, the prepared slides are air dried and then stained by May-Grunwald stain, followed by a Giemsa solution stain. The stained slides are rinsed with deionized water, air dried, and rinsed with methanol. Then the slides are placed in xylene for clearing. Finally, they are mounted and then analyzed (1000 cells are scored for each sample).39?41
After sacrificing animals, cells can be isolated from lung tissue. The inferior vena cava is severed, and then the lung tissue is perfused through the right ventricle with ethy-leneglycoltetraacetic acid (EGTA) solution in Hanks' balanced salt solution (HBSS). After perfusing until it is blanched, the lung tissue is then inflated through the trachea with solution containing trypsin, EDTA, and collagenase. The lung tissue is removed, minced, and incubated with rocking in EDTA containing enzyme solution. The supernatant containing individual cells is collected, and then DNase I is added. After centrifugation of the cells, the pellet is washed with complete medium. Aliquouts of 1×106 cells in 2 ml complete medium are seeded onto square cover glass in a tissue culture dish. To prevent the cells from cytokinesis, an inhibitor of the mitotic spindle namely cytochalasin B (Cyt-B) is added to each culture after 24 hours of the culture, allowing distinguish cells that have completed one nuclear division and consequently become binucleated. After 48 hours of adding Cyt-B, the seeded cells are then fixed with 3:1 methanol:acetic acid or 100% ethanol for staining. The cells are stained with Giemsa solution for calculating the frequency of binucleate cells having MN.42,43
In spite of regulatory directives concerning the reduction of animal use in safety test, modifications to genotoxicity testing guidelines recently offer the utility of two
Genotoxic events have been regarded as a crucial stage in the initiation of carcinogenesis.44 Genomic instability may enable a cell to accumulate stable genome mutations, and it represents an early step in the carcinogenesis.4 When cells having modified DNA and abnormal genome continually survive, the abnormal cell can be a latent cancer cell or it can give rise to a cancer.45 Most cancers have shown many chromosomal aberrations, and these alterations can be detected in both benign and malignant tumors.2,4 Relying on the standard genotoxicity and/or carcinogenicity tests including
For a more comprehensive investigation of genotoxicity in animal models, the National Toxicology Program (NTP) has performed a combined assay using
The genotoxicity assays have been used for studying the carcinogenesis of chemicals and assessing the potential carcinogenicity of chemicals to humans.44 The findings based on comet assay and MN assay have shown correlations between genotoxicity and preneoplastic/neoplastic changes. The
Few studies have been performed to investigate the quantitative dose-response relationship between carcinogenesis and
As mentioned above, the comet and MN assay-based-analysis would enable the prediction of the potential carcinogenicity of diverse substances including chemical and physical agents via carcinogenicity databases. Due to flexibility feature, the
According to several studies and national/international institution guidelines, integrating the
Potential carcinogenicity studies using
Chemical | Species | Tissue | Route | Dose | Sampling time | Result of the Assays | Carcinogenicity Data | Reference |
---|---|---|---|---|---|---|---|---|
1,2-DMH 2HCl | F344 rat | Liver | po | 100, 200 mg/kg | 3, 4, 5 days | Positive at 3, 4, 5 days treatment (MN assay) | IARC: 2A Carcinogenicity: + | [54] |
1,4-Dioxane | CD-1 mouse | Liver | po | 1,500, 2,500, 3,500 mg/kg/day for 5 days | 24 h | Positive in 2,500, 3,500 mg/kg (MN assay) | IARC: 2B Carcinogenicity: + | [55] |
1,4-Dioxane | CD-1 mouse | Liver | po | 1,000, 2,000, 3,000 mg/kg | 6 days | Positive in 2000mg/kg (MN assay) | IARC: 2B Carcinogenicity: + | [56] |
2-Acetylaminofluorene | SD rat | Bone marrow, peripheral blood | po | 125, 500 mg/kg ×2 days | 24 h | Positive in bone marrow, blood at two doses (MN assay) | IARC: nd Carcinogenicity: + | [57] |
2-Acetylaminofluorene | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 400 mg/kg | 3, 24 h | Positive in liver and kidney 3 h after treatment (comet assay) | IARC: nd Carcinogenicity: + | [57] |
2,4-Diaminotoluene | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 240 mg/kg | 3, 24 h | Positive in liver and kidney 3, 24 h after treatment and in lung 3 h after treatment (comet assay) | IARC: 2B Carcinogenicity: + | [58] |
2,4-dinitrotoluene | F344 rat | Liver | po | 200, 400 mg/kg | 3, 4, 5 days | Positive (MN assay) | IARC: 2B Carcinogenicity: + | [54] |
2,4-dinitrotoluene | F344 rat | Liver | po | 75, 150, 300 mg/kg (two-dose assay) | 4 days | Positive in 75, 150, 300 mg/kg (MN assay) | IARC: 2B Carcinogenicity: + | [57] |
2,6-dinitrotoluene | F344 rat | Liver | po | 50, 100, 200 mg/kg (two-dose assay) | 4 days | Positive in 50, 100, 200 mg/kg (MN assay) | IARC: 2B Carcinogenicity: + | [57] |
Acrylonitrile | SD rat | Bone marrow, peripheral blood | iv | 124.8, 125 mg/kg × 2 days | 24 h | Positive in bone marrow but not in blood (MN assay) | IARC: 2B Carcinogenicity: + | [57] |
Arsenic acid solution | CD-1 mouse | Bone marrow | ip | 1, 5, 10, 20 mg/kg/d for 4 days | 24 h | Positive in 10, 20 mg/kg/day (MN assay) | IARC: 1 Carcinogenicity: + | [59] |
Atrazine | Wistar rat | Liver, blood | po | 300 mg/kg/d for 7, 14, 21 days | Not described | Positive at periods of 7, 14, and 21 days (comet assay, MN assay) | IARC: 3 Carcinogenicity: + | [39] |
Auramine | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 80 mg/kg | 3, 24 h | Positive in liver, kidney, and lung 3 h after treatment (comet assay) | IARC: 2B Carcinogenicity: + | [58] |
Benzene | NMRI mice | Blood lymphocytes, bone marrow | po | 40, 200, 450 mg/kg | 6 h | Positive in lymphocytes at 200, 450 mg/kg and in bone marrow at 40, 200, 450 mg/kg (comet assay) | IARC: 1 Carcinogenicity: + | [60] |
Benzo[α]pyrene | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | po | 250 mg/kg | 3, 24 h | Positive in liver and lung 3 h after treatment (comet assay) | IARC: 2A Carcinogenicity: + | [56] |
Cadmium chloride | White swiss mouse | Bone marrow | ip | 1.9, 5.7, 7.6 mg/kg | 24 h | Positive in 1.9, 5.7, 7.6 mg/kg (MN assay) | IARC: 1 Carcinogenicity: + | [54] |
Chloroform | SD rat | Kidney | po | 4 mmol/kg | 2 days | All agents are positive in kidney (MN assay) | IARC: 2B Carcinogenicity: + | [60] |
Dimethylnitrosamine | F344 rat | Liver | po | 2.5, 5, 10 mg/kg (two-dose assay) | 3, 4, 5 days | Positive in 5, 10 mg/kg (MN assay) | IARC: 2A Carcinogenicity: + | [54] |
Ethylene thiourea | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 2,000 mg/kg | 3, 24 h | Positive in liver, kidney, lung, and spleen 3, 24 h after treatment (comet assay) | IARC: 3 Carcinogenicity: + | [58] |
Lambda cyhalothrin | Wistar rat | Bone marrow | po | 0.8, 3.06, 6.12 mg/kg | 30 h | Positive in all doses (MN assay) | IARC: nd Carcinogenicity: + | [61] |
Mitomycin C | F344 rat | Liver | ip | 0.5, 1, 2 mg/kg (two-dose assay) | 4 days | Positive in 0.5, 1, 2 mg/kg (MN assay) | IARC: 2B Carcinogenicity: + | [57] |
BALB/c mouse | Stomach | po | 100 mg/kg | 3, 4 days | Positive in stomach 3, 4 days after injection (MN assay) | IARC: 2A Carcinogenicity: + | [56] | |
BALB/c mouse | Peripheral blood | po | 100 mg/kg | 2, 3 days | Positive in peripheral blood 2, 3 days after injection (MN assay) | IARC: 2A Carcinogenicity: + | [56] | |
CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 200 mg/kg | 3, 24 h | Positive in liver, kidney, and spleen 3 h after treatment. Positive in all tissues 24 h after treatment (comet assay) | IARC: 2B Carcinogenicity: + | [58] | |
CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 2,000 mg/kg | 3, 24 h | Positive in liver and spleen 3 h after treatment (comet assay) | IARC: 2B Carcinogenicity: + | [58] | |
Phenobarbital | SD rat | Liver | po | 30, 90, 120 mg/kg/day for 3 days. | 3 h | Positive at 120 mg/kg/d for 3 days, but not at all doses for 29 days (comet assay) | IARC: 2B Carcinogenicity: + | [62] |
Potassium chromate(VI) | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 80 mg/kg | 3, 24 h | Positive in liver and lung 3 h after treatment (comet assay) | IARC: 1 Carcinogenicity: + | [4] |
Quinoline | F344 rat | Liver | po | 30, 60, 90 mg/kg (two-dose assay) | 3, 4, 5 days | Positive in 60, 90 mg/kg (MN assay) | IARC: nd Carcinogenicity: + | [58] |
Styrene-7,8-oxide | CD-1 mice | Liver, kidney, lung, spleen, bone marrow | ip | 400 mg/kg | 3, 24 h | Positive in all tissues 3 h after treatment (comet assay) | IARC: 2A Carcinogenicity: + | [63] |
Thioacetamide | C57BL/6 Mouse | Bone marrow | po | 375?1,500 mg/kg | 1, 2 days | Positive (MN assay) | IARC: 2B Carcinogenicity: + | [64] |
Trichloroethylene | SD rat | Kidney | po | 4 mmol/kg | 2 days | All agents are positive in kidney (MN assay) | IARC: 1 Carcinogenicity: + | [64] |
Thiabendazole | ddy mouse | Stomach, liver, kidney, bladder, brain, bone marrow, lung | po | 10, 100, 200 mg/kg | 3, 24 h | Positive in all organs at 200 mg/kg, 3 h treatment | IARC: nd Carcinogenicity: + | [8] |
i Ip, Intraperitoneally; Po, Oral administration; Iv, Intravenous; Nd, not determined; IARC 1, Carcinogenic to humans; IARC 2A, Probably carcinogenic to humans; IARC 2B, Possibly carcinogenic to humans; IARC 3, Not classifiable as to its carcinogenicity to humans; IARC 4, Probably not carcinogenic to humans..
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