Preclinical Development Handbook || In Vitro Mammalian Cytogenetic Tests

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    R. Julian Preston National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

    Preclinical Development Handbook: Toxicology, edited by Shayne Cox GadCopyright 2008 John Wiley & Sons, Inc.


    6.1 Purpose of Cytogenetic Tests 6.2 History of Cytogenetic Testing Protocols 6.3 Description of Endpoints

    6.3.1 Chromosomal Structural Alterations 6.3.2 Chromosomal Numerical Alterations (Aneuploidy) 6.3.3 Sister Chromatid Exchanges (SCEs) 6.3.4 Micronuclei

    6.4 Description of Cell Test Systems 6.4.1 Established Cell Lines 6.4.2 Human Lymphocyte Cultures

    6.5 Requirements for Cytogenetic Assays 6.6 Description of Assays

    6.6.1 Cell Growth and Maintenance 6.6.2 Metabolic Activation 6.6.3 Control Groups 6.6.4 Test Chemicals 6.6.5 Exposure Concentrations 6.6.6 Treatment with Test Substances 6.6.7 Culture Harvest Times 6.6.8 Chromosome Preparations 6.6.9 Analysis of Cytogenetic Alterations 6.6.10 Statistical Analysis and Evaluation of Results 6.6.11 Test Conclusions 6.6.12 Test Report

    6.7 Future Directions Acknowledgments References



    The fi rst step in any cancer risk assessment process for exposures to environmental chemicals is hazard identifi cation . Thus, in order for new products to be registered for the fi rst time or to be reregistered, an assessment of whether or not they present a potential hazard has to be conducted. This applies equally to products for agricul-tural, pharmaceutical, or consumer use. In the present context, a hazard is con-sidered to be represented by genetic alterations assessed as gene mutations or chromosomal alterations. In particular, this chapter concentrates on the induction of chromosomal alterations, both numerical and structural ones.


    The analysis of chromosomal alterations in cells exposed to environmental agents, particularly ionizing radiation, has a long history, starting in a quantitative fashion with the pioneering work of Karl Sax [1] using pollen grains of Tradescantia . These studies provided information on the dose response for X - ray - and neutron - induced chromosome aberrations and the relative effectiveness of acute, chronic, and frac-tionated exposures. These types of studies were expanded over the next decade or so to include the assessment of the cytogenetic effects produced by chemicals. The types of assay available for cytogenetic analysis were greatly expanded by the ability to grow mammalian cells in vitro , either as primary cultures or as transformed cell lines. This capability was further enhanced to include the use of readily available human cells following development of the in vitro culture of human lymphocytes by Moorhead et al. [2] . This method relied on the use of phytohemagglutinin to stimulate peripheral lymphocytes to reenter the cell cycle, thereby allowing for metaphase cells to be obtained. At this time, it was necessary to obtain metaphase cells because all cytogenetic assays relied on microscopic evaluation of chromo-somes at metaphase. The use of colcemid to inhibit cells at metaphase and of hypotonic solutions to swell the cells for easy analysis of chromosomal alterations also made cytogenetic tests much more reliable, repeatable, and technically straight-forward [3] .

    The standardization of approaches for visualizing chromosomes for assessing structural and numerical alterations following exposure to radiation or chemicals led to the development of cytogenetic assays for testing chemicals for potential hazard to humans. A plethora of different test systems became available, and there was unique merit to many of these. With fairly extensive validation studies, the assays that were considered to be the most straightforward to conduct and provided the more reliable predictions of clastogenicity (chromosome breakage) for carcino-gens and noncarcinogens were incorporated into testing guidelines. This history is concisely described in Chapter 1 of the Evaluation of Short - Term Tests for Carcino-gens: Report of the International Collaborative Program [4] . These in vitro cytoge-netic assays incorporate chromosome alterations, sister chromatid exchanges (SCEs), or micronuclei as endpoints, and permanent cell lines (especially Chinese hamster) as the test cells. Based on the selection of the highest performing assays as regards carcinogen/noncarcinogen predictions, a number of national and international orga-nizations developed test batteries that could further enhance predictive value. Typi-

  • cally, these include a bacterial reverse mutation assay, an in vitro cytogenetic assay in mammalian cell cultures and/or an in vitro gene mutation assay in mammalian cell cultures, and an in vivo cytogenetic assay in rodent bone marrow cells.

    This standard battery of genotoxicity tests can satisfy the requirements of various global regulatory bodies. For example, several national and international regulatory agencies have presented minimum data requirements for regulatory reviews of commercial chemicals, and these include the use of a standard battery of genotoxic-ity tests as described above [5] . Although there have been some modifi cations to these basic assays for improving overall sensitivity and predictivity, the basic prin-ciples underlying the assays remain the same, as discussed in Sections 6.5 and 6.6 .

    Recent advances in chromosomal imaging techniques, based on the development of sophisticated fl uorescence in situ hybridization (FISH) methods, have signifi -cantly enhanced the understanding of the mechanisms of chromosome aberration induction and the ability to identify specifi c aberration types (e.g., reciprocal translocations). These techniques have generally remained in the purview of mecha-nistic studies and have not been incorporated into cytogenetic tests for assessing carcinogens.


    6.3.1 Chromosomal Structural Alterations

    A broad range of structural alterations can be observed in metaphase cells using either conventional cytogenetic techniques or fl uorescence in situ hybridization (FISH). The types of structural alterations produced by treatment with chemicals and radiation are the same as those observed in untreated cells. Thus, treatment with a clastogenic agent enhances the frequencies of chromosomal alterations but does not produce a different spectrum of types. A comprehensive description of the classes of structural chromosomal alterations can be found in Savage [6] . In general terms, structural alterations can be subdivided into unstable (generally nontransmis-sible) and stable (transmissible to daughter cells at division). Within these two broad categories, an important distinction is made between chromosomal alterations pro-duced prior to DNA replication (G 1 ) and those produced during (S) or after (G 2 ) replication. The former are called chromosome - type aberrations because they involve both chromatids of a chromosome and the latter are called chromatid - type because they involve one of the two chromatids. An exception is the so - called iso-chromatid aberration that is produced in S or G 2 of the cell cycle but involves both chromatids. Within each of these two classes, chromosomal alterations can be described as deletions (loss of chromosomal material) and exchanges (between or within chromosomes). Deletions are generally unstable alterations and exchanges can be stable (reciprocal exchanges) or unstable (dicentrics). For the purposes of most mammalian cytogenetic tests that are used for hazard identifi cation, this level of defi nition of the range of structural alterations is generally suffi cient.

    6.3.2 Chromosomal Numerical Alterations (Aneuploidy)

    There are chemicals that can alter the normal process of cell division either resulting in chromosomes failing to separate at metaphase or preventing one or more



    chromosomes attaching to the mitotic spindle. The results are that daughter cells will have either additional chromosomes beyond the normal diploid number (hyper-ploidy) or fewer chromosomes than the diploid number (hypoploidy). Because of possible artifacts produced during the preparative stages of cells for cytogenetic analysis that can result in chromosome loss, the general approach for assessing aneuploidy is to place greater emphasis on hyperploidy.

    6.3.3 Sister Chromatid Exchanges ( SCE s )

    During the course of DNA replication, errors can occur that lead to the reciprocal exchange between the two sister chromatids of a chromosome. The result is the so - called sister chromatid exchange. While such exchanges had been identifi ed by genetic methods for decades and using radioisotopes in the 1950s, it was not until the 1970s that a simple cytogenetic method was developed for direct observation of SCE [7, 8] . This method relied in general terms on differential staining of the sister chromatids one being lightly stained with Giemsa and the other darkly stained. Exchanges between the sister chromatids could readily be seen in metaphase cells as light dark switches. The principle behind the method was the preferential elution of DNA from one chromatid based on incorporation of bromodeoxyuridine (BrdU), binding of a BrdU - sensitive fl uorescence dye to asymmetrically BrdU - labeled chromatids, and the subsequent preferential breakage of the DNA containing the dye by fl uorescent light and its elution by warm saline sodium citrate. The chromatid with more DNA stains dark with Giemsa and that with less DNA stains lighter [7] . More recently, fl uorescent antibodies to BrdU have been used for sister chromatid differentiation [9] .

    It has been demonstrated that a wide range of chemicals can increase the fre-quency of SCEs in a broad range of cell types [10] . There is a background level of SCEs that varies with cell type and concentration of BrdU; these SCEs are a con-sequence of the BrdU incorporation into the DNA. The analysis of SCEs can be a useful component of hazard identifi cation, although it needs to be noted that there is no adverse phenotypic outcome known to be associated with the induction of SCEs. There is, however, a high frequency of SCEs in cells from individuals with Bloom syndrome that is a refl ection of error - prone DNA replication of the BrdU - containing template [11] . Thus, SCE increases probably refl ect an enhancement of errors in DNA replication, induced, for example, by a clastogen that produces DNA adducts. An advantage of the SCE assay is its high level of sensitivity of detecting chromosomal effects that are produced by S - phase - dependent mechanisms.

    6.3.4 Micronuclei

    The analysis of structural chromosomal alterations by standard metaphase analysis requires a high level of expertise and is time consuming. For hazard identifi cation purposes, the analysis of micronuclei can be equally informative. Micronuclei are membrane - bounded structures that form either from acentric pieces of DNA (dele-tions) or whole chromosomes that are not incorporated into daughter nuclei. It is possible to distinguish between acentric fragments and whole chromosomes using an antibody to the kinetochore component of the centromere; a positive stain rep-resents a micronucleus containing a whole chromosome [12] . Thus, the micronucleus

  • assay can be used for the assessment of structural and numerical chromosomal alterations. While the micronucleus assay does not provide the detail for all types of chromosomal alterations induced by a clastogen that would be obtained by meta-phase analysis, it is a robust assay for the identifi cation of a clastogen.


    6.4.1 Established Cell Lines

    It is possible to use any permanent cell line that is cycling for cytogenetic tests. However, it is considerably easier for analysis of the data to use cell lines that have a stable karyotype that is close to the diploid number for the particular species. Chinese hamster cell lines (CHO and V79) have frequently been used because of their low near - diploid number of chromosomes (2 n is normally 22). Primary human cell lines or virally transformed ones can be used, but they have a relatively low mitotic index that makes analysis more burdensome than with rapidly dividing cells. Thus, any cell line with a stable karyotype and a high mitotic index is most suitable for cytogenetic tests. Human lymphocytes can be virally transformed to produce lymphoblastoid cell lines that generally have a stable karyotype close to the diploid number, but have the advantage of a relatively high mitotic index.

    6.4.2 Human Lymphocyte Cultures

    Human peripheral blood lymphocytes are nondividing cells but can be stimulated to reenter the cell cycle by use of a mitogen such as phytohemagglutinin. The stimu-lated cells move somewhat synchronously through the fi rst cell cycle, so that cells can be treated in G 1 , S, and/or G 2 either by selection of the appropriate treatment time after stimulation or by selection of an appropriate time after treatment for cell sampling and fi xation. Mitogen stimulated cells have a relatively high mitotic index, especially at the fi rst metaphase, and being primary cultures they have a stable diploid karyotype when sampled from a normal individual. A concern with their routine use is that blood samples have to be obtained from suitable volunteers and this requires some form of informed consent. However, the process for meeting the consent requirement can quite readily be met.


    While it is possible to use any cycling cell system for cytogenetic analysis, it is highly advantageous to use one that has a stable diploid or near - diploid karyotype. Again, as noted previously, a high mitotic index is also greatly advantageous, if nothing else because this reduces the time to search for analyzable metaphase cells. Primary human lymphocyte cultures are very suitable for meeting both of these needs.

    The great majority of chemical clastogens produce structural aberrations by inducing errors of DNA replication (e.g., from an induced DNA adduct) and so treatment with a test chemical has to be for cells in the S phase or that pass through the S phase between treatment and analysis at metaphase [13] . For maximum sen-sitivity, treatment in S phase is required such that repair of induced DNA damage


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