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. In vivo genotoxicity tests using tissues can be used when obtaining in vitro positive results, that can reflect absorption, excretion, distribution, and metabolism of chemicals but the in vitro test does not.^7,8 The in vitro assay has been considered as a genotoxicity test for screening substances (e.g. drug candidates, medicinal plant extract, chemical substances, etc.) and evaluating their initial safety while the in vivo assay provides detailed information of biological and physiological significance. It can determine whether any potential mutagenic effects that have shown in the in vitro step have appeared again in the animal’s whole physiological system.⁵ Consequently, the in vivo assays have been known as important processes in the verification of in vitro test and risk assessments for humans, indicating that they have more impact than in vitro assays.

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 in vivo comet assay used to detect the genotoxic potential of chemicals has been recognized as a second in vivo genotoxicity assay by the International Conference on Harmonization (ICH- S2 (R1)) guidance (2012) with in vivo micronucleus (MN) assay. In the field of genotoxicology, the in vivo comet assay has been considered as powerful tools to distinguish between genotoxic carcinogens and nongenotoxic carcinogens as well as to identify carcinogens and mutagens.¹⁰ Consequently, the comet assay can be a biomarker for detecting both genetic susceptibility and the DNA damage related to carcinogenesis.^11,12 Most carcinomas normally show a greater degree of DNA damage with extensive comet tails than that found in the tissue cells from controls.¹³ In several researches, CD-1 mouse strain has been commonly used as standard animal model in the in vivo comet assay.

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 in vivo comet assay with the in vivo MN assay.²¹

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 in vivo comet assay and in vivo MN assay for testing genotoxicity and predicting carcinogenic potential (Fig. 1). By comparing potential carcinogenicity studies using genotoxicity assays with other carcinogenicity databases, prediction of carcinogenic potential will be discussed in the following section and the development of both in vivo assays of comet assay and micronucleus assay will be further warranted.

In vivo comet assay has been generally performed to detect DNA strand breaks as the comet tail-like-shapes formed by DNA fragment in cells. In principle, after the lysis and electrophoresis steps of cells, the negatively charged DNA fragments migrate out of the cell toward the anode, and appear in a comet shape visualized under a fluorescence microscope.²⁴ In a testing for genotoxic carcinogen agents, increased migration of the negatively charged DNA fragments toward the anode indicate increased the numbers of DNA strand breaks. Alkaline version of comet assay was introduced for the detection of double strand breaks, single strand breaks, alkali labile sites, DNA cross-linking, and incomplete excision repair sites.^10,24 The alkaline-based comet assay has shown increased sensitivity for detecting genotoxic agents because most genotoxic agents may induce more single strand breaks or alkali labile sites than double strand breaks.²⁵ Overall, the advantages of the comet assay in comparison to other genotoxicity assays include: (1) applicability to various tissues and cell types (flexibility), (2) no number of cells requirement, (3) sensitivity to detecting DNA damage, (4) brevity of performance time, and (5) the relatively low cost of the method.^10,20,26?²⁹ Prior to the establishment of an OECD test guideline, the in vivo comet assay using tissues was established by JaCVAM validation studies.

Any type of animal tissue (e.g. liver, stomach, and blood cells) can be applied to the in vivo comet assay, as long as the tissue types can provide high-quality single cell suspension.³⁰ This methodology of comet assay refers to JaCVAM validation studies (Fig. 2).³¹

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 in vivo MN assay was established by the OECD guidelines, and has been continuously being developed through many studies.

The in vivo MN assay is known as simple and sensitive screening method of clastogenic agents in the target tissues (e.g. blood and lung sample). In addition, many studies of in vivo MN assays using tissues have been so far published (Fig. 3).³⁷

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 in vivo genotoxicity assays of comet and MN as a follow-up to an in vitro positive.⁶ Both in vivo assays can be achieved into one informative investigation. Combining these two assays with individual difference in sensitivity, measurable endpoints, and types of parameter analyzed significantly potentiate the current standard capabilities and performance for assessing genotoxicity as well as for evaluating carcinogenesis incidence and safety risk without requirement of additional animals.

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 in vivo comet and MN assay, the International Agency for Research on Cancer (IARC) has published lists of agents that can cause cancer in humans and has provided reliable data on human carcinogenicity.⁴⁴Table 1 presented the results of the in vivo comet and MN assay that have investigated the genotoxicity of potential carcinogens classified by the IARC in carcinogenicity database. Consequently, the genotoxicity results may enable researchers to predict the carcinogenic potential of chemicals (Table 1).

For a more comprehensive investigation of genotoxicity in animal models, the National Toxicology Program (NTP) has performed a combined assay using in vivo comet and MN assay.⁴⁶ As the comet assay has been known to detect most carcinogens that have been identified as equivocal or negative in the MN assay, the results of both assays are considerably regarded as complementary issue. Therefore, the combined in vivo genotoxicity assay has been recommended to investigate genotoxic potential in several recent studies.⁴⁶?⁴⁸ In addition, the combined in vivo genotoxicity assay has been regarded as a useful methodology for evaluation of genetic hazard in safety risk assessment because it offers two distinct genotoxicity endpoints. Based on the advantages of the combined in vivo genotoxicity assay, the NTP has introduced this combined assay as a part of detecting the genotoxicity of substances with public health concern.⁴⁶ In both in vivo comet and MN assay, mouse strains of BALB/c, C57BL/6, CD-1, ddy, NMRI and White Swiss, and rat strains of F344, SD, and Wistar have been extensively used. In particular, CD-1 mouse strain has been mainly used in the comet assay while strains of F344 rat and SD rat have been frequently used in the MN assay.

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 in vivo comet assay studies have been performed to assess genotoxicity in terms of cancer development.⁷ In addition, correlations between MN induction and cancer development have been reported in several studies.⁴⁹ Indeed, MN has been known as a manifestation of chromosomal instability that occurs in cancer.³⁵ Numerous studies focused on the application of MN have shown a significant increase of the MN frequency in peripheral blood lymphocytes of cancer patients in comparison with healthy control patients.⁵⁰ These studies have suggested that increased results of the MN frequency are related with an early step in carcinogenesis.⁵¹

Few studies have been performed to investigate the quantitative dose-response relationship between carcinogenesis and in vivo genotoxicity results. Among them, one study was conducted to evaluate their quantitative relation. The quantitation utilized the lowest effective dose (LED) derived from the in vivo genotoxicity tests (MN, comet, sister chromatid exchange, and chromosome aberration test) and the T25 as the chronic daily dose (mg/kg/day that induces tumor in 25% at specific tissue) from rodent carcinogenicity studies. The relation between the LED and T25 was identified as a linear correlation.^52,53 In addition, another study utilized the benchmark dose (BMD) to evaluate the quantitative relation among them. The BMD-based approach has been used for correlating equipotent doses of carcinogenicity studies with equipotent doses of in vivo genotoxicity studies. Likewise, positive relation between carcinogenicity and in vivo genotoxicity results were identified using the BMD values. The relation between the lowest BMD10 of MN and the tissue-matched carcinogenicity BMD10 was evaluated, and thus quantitative correlation was observed.⁵²

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 in vivo comet assay can be incorporated into most standard testing batteries to provide supplemental data of target tissue without requirement of additional duration or resources in an independent experimentation setting. To further achieve appropriateness of study design and accuracy of comet data interpretation, the recognition of significant discrepancy between the comet assay and the MN assay are necessary for the combination of either the comet assay or the MN assay with standard genotoxicity tests and/or toxicological studies. Herewith, the combined comet and MN assay protocol has proven to be a sensitive and efficient for evaluating within the same animals toward multiple classes of genotoxic agents across a wide range of target tissues. This approach takes advantage of the minimal use of animal number. Taken together, these recommendations provide an effective methodology for combining the in vivo comet and MN assays and for interpreting assay data. This approach would improve safety testing of critical target tissue data with increased sensitivity while minimizing animal use, reducing exposure times and toxicity. Further investigations should be optimized for these two assays to take full advantage of the increased sensitivity, capability, and flexibility.

According to several studies and national/international institution guidelines, integrating the in vivo comet and in vivo MN assays has been successfully performed for follow-up testing of positive in vitro results. They may be served as potential tool for the assessment of local gentoxicity, especially for tissues or cell types which cannot be easily measured with other standard testing methods. Several results of these two in vivo genotoxicity assays may indicate that the genotoxicity has positive correlation with the carcinogenicity. Such approach-based findings will provide practical consequences in the risk assessment processes and further development of substances. In many studies, the two in vivo genotoxicity assays have been extensively used to investigate the chemicals classified by IARC, and the genotoxicity assay results indicate that the carcinogenicity of potent substances can be predicted by the in vivo comet and in vivo MN assays. Therefore, the combined in vivo genotoxicity assay may be used to detect the carcinogenic potential of substances for the prevention of cancer. Such approach-based findings will provide practical consequences in the risk assessment processes and further development of substances.