Introduction to Assay Design
Genetic toxicology assays serve two interrelated but distinct purposes in the toxicologic evaluation of chemicals: (1) identifying mutagens for purposes of hazard identification and (2) characterizing dose–response relationships and mutagenic mechanisms.
Table 9–1 lists many of the assays employed in genetic toxicology. Some assays for gene mutations detect forward mutations, whereas others detect reversion. Forward mutations are genetic alterations in a wild-type gene and are detected by a change in phenotype caused by the alteration or loss of gene function. In contrast, a back mutation or reversion is a mutation that restores gene function in a mutant and thereby brings about a return to the wild-type phenotype. The simplest gene mutation assays rely on selection techniques to detect mutations. By imposing experimental conditions under which only cells or organisms that have undergone mutation can grow, selection techniques greatly facilitate the identification of rare cells that have experienced mutation among the many cells that have not.
Table 9–1 Overview of genetic toxicology assays. ||Download (.pdf)
Table 9–1 Overview of genetic toxicology assays.
I. DNA damage and repair assays
A. Direct detection of DNA damage
- Alkaline elution assays for DNA strand breakage
- Comet assay for DNA strand breakage
- Assays for chemical adducts in DNA
B. Bacterial assays for DNA damage
- Differential killing of repair-deficient and wild-type strains
- Induction of the SOS system by DNA damage
C. Assays for repairable DNA damage in mammalian cells
- Unscheduled DNA synthesis (UDS) in rat hepatocytes
- UDS in rodent hepatocytes in vivo
II. Prokaryote gene mutation assays
A. Bacterial reverse mutation assays
- Salmonella/mammalian microsome assay (Ames test)
- E. coli WP2 tryptophan reversion assay
- Salmonella specific base-pair substitution assay (Ames-II assay)
- E. colilacZ specific reversion assay
B. Bacterial forward mutation assays
- E. coli lacI assay
- Resistance to toxic metabolites or analogs in Salmonella
III. Assays in nonmammalian eukaryotes
A. Fungal assays
- Forward mutations, reversion, and small deletions
- Mitotic crossing over and gene conversion in yeast
- Mitotic aneuploidy: chromosome loss or gain in yeast
- Meiotic nondisjunction in yeast or Neurospora
B. Plant assays
- Gene mutations affecting chlorophyll in seedlings or waxy in pollen
- Tradescantia stamen hair color mutations
- Chromosome aberrations or micronuclei in mitotic or meiotic cells
- Aneuploidy detected by pigmentation or cytogenetics
C. Drosophila assays
- Sex linked recessive lethal test in germ cells
- Heritable translocation assays
- Sex chromosome loss tests for aneuploidy
- Induction of mitotic recombination in eyes or wings
IV. Mammalian gene mutation assays
A. In vitro assays for forward mutations
- tk mutations in mouse lymphoma or human cells
- hprt or xprt mutations in Chinese hamster or human cells
B. In vivo assays for gene mutations in somatic cells
- Mouse spot test (somatic cell specific locus test)
- hprt mutations (6-thioguanine-resistance) in rodent lymphocytes
C. Transgenic assays
- Mutations in the bacterial lacI gene in “Big Blue®” mice and rats
- Mutations in the bacterial lacZ gene in the “Muta™ Mouse”
- Mutations in the phage cII gene in lacI or lacZ transgenic mice
- Point mutations and deletions in the lacZ plasmid mouse
- Point mutations and deletions in delta gpt mice and rats
V. Mammalian cytogenetic assays
A. Chromosome aberrations
- Metaphase analysis in cultured Chinese hamster or human cells
- Metaphase analysis of rodent bone marrow or lymphocytes in vivo
- Cytokinesis-block micronucleus assay in human lymphocytes
- Micronucleus assay in mammalian cell lines
- In vivo micronucleus assay in rodent bone marrow or blood
C. Sister chromatid exchange
- SCE in human cells or Chinese hamster cells
- SCE in rodent tissues, especially bone marrow
D. Aneuploidy in mitotic cells
- Mitotic disturbance seen by staining spindles and chromosomes
- Hyperploidy detected by chromosome counting
- Chromosome gain or loss in cells with intact cytoplasm
- Micronucleus assay with centromere labeling
- Hyperploid cells in vivo in mouse bone marrow
- Mouse bone marrow micronucleus assay with centromere labeling
VI. Germ cell mutagenesis
A. Measurement of DNA damage
- Molecular dosimetry based on mutagen adducts
- UDS in rodent germ cells
- Alkaline elution assays for DNA strand breaks in rodent testes
B. Gene mutations
- Mouse specific-locus test for gene mutations and deletions
- Mouse electrophoretic specific-locus test
- Dominant mutations causing mouse skeletal defects or cataracts
- Mouse tandem-repeat loci analysis
C. Chromosomal aberrations
- Cytogenetic analysis in oocytes, spermatogonia, or spermatocytes
- Micronuclei in mouse spermatids
- Mouse heritable translocation test
D. Dominant lethal mutations
- Mouse or rat dominant lethal assay
- Cytogenetic analysis for aneuploidy arising by nondisjunction
- Sex chromosome loss test for nondisjunction or breakage
- Micronucleus assay in spermatids with centromere labeling
- FISH with probes for specific chromosomes in sperm
Studying mutagenesis in intact animals requires more complex assays, which range from inexpensive short-term tests that can be performed in a few days to complicated assays for mutations in mammalian germ cells. Typically, there remains a gradation in which an increase in relevance for human risk entails more elaborate and costly tests.
Many compounds that are not themselves mutagenic or carcinogenic can be activated into mutagens and carcinogens by mammalian metabolism. Such compounds are called promutagens and procarcinogens. The most widely used metabolic activation system in microbial and cell culture assays is a postmitochondrial supernatant from a rat liver homogenate, along with appropriate buffers and cofactors. Most of the short-term assays in Table 9–1 require exogenous metabolic activation to detect promutagens. Exceptions are those in intact mammals.
Despite their usefulness, in vitro metabolic activation systems cannot mimic mammalian metabolism perfectly. There are differences among tissues in reactions that activate or inactivate foreign compounds, and organisms of the normal flora of the gut can contribute to metabolism in intact mammals.
DNA Damage and Repair Assays
Some assays measure DNA damage itself rather than mutational consequences of DNA damage. They may do so directly, through such indicators as chemical adducts or strand breaks in DNA, or indirectly, through measurement of biological repair processes. Adducts in DNA can be detected by 32P-postlabeling, immunologic methods using antibodies against specific adducts, or fluorometric methods in the case of such fluorescent compounds.
A rapid method of measuring DNA damage is the comet assay. In this assay, cells are incorporated into agarose on slides, lysed so as to liberate their DNA, and subjected to electrophoresis. The DNA is stained with a fluorescent dye for observation and image analysis. Because broken DNA fragments migrate more quickly than larger pieces of DNA, a blur of fragments (a “comet”) is observed when the DNA is extensively damaged. The extent of DNA damage can be estimated from the length and other attributes of the comet tail. The comet assay appears to be a sensitive indicator of DNA damage with broad applicability among diverse species, including plants, worms, mollusks, fish, and amphibians.
The occurrence of DNA repair can serve as a readily measured indicator of DNA damage. The most common excision repair assay in mammalian cells is an assay for unscheduled DNA synthesis (UDS). The occurrence of UDS indicates that the DNA had been damaged.
Gene Mutations in Prokaryotes
The most common means of detecting mutations in microorganisms is selecting for reversion in strains that have a specific nutritional requirement differing from wild-type members of the species; such strains are called auxotrophs. In the Ames assay, one measures the frequency of histidine-independent bacteria that arise in a histidine-requiring strain in the presence or absence of the chemical being tested. Auxotrophic (nutrient-deficient) bacteria are treated with the chemical of interest and plated on medium that is deficient in histidine; if the colony survives, it must have a reversion mutation that allows it to survive without exogenous histidine.
The development of specific reversion assays of histidine mutations in Salmonella strains and of lacZ mutations in Escherichia coli has made the identification of specific base-pair substitutions more straightforward.
Genetic Alterations in Nonmammalian Eukaryotes
Gene Mutations and Chromosome Aberrations
The fruit fly, Drosophila, has long occupied a prominent place in genetic research because of the sex-linked recessive lethal (SLRL) test. The SLRL test permits the detection of recessive lethal mutations at 600 to 800 different loci on the X chromosome by screening for the presence or absence of wild-type males in the offspring of specifically designed crosses. A significant increase over the frequency of spontaneous SLRLs in the lineages derived from treated males indicates mutagenesis. The SLRL test yields information about mutagenesis in germ cells, which is lacking in microbial and cell culture systems.
Genetic and cytogenetic assays in plants continue to find use in special applications, such as in situ monitoring for mutagens and exploration of the metabolism of promutagens by agricultural plants. In situ monitoring entails looking for evidence of mutagenesis in organisms that are grown in the environment of interest.
Assays in nonmammalian eukaryotes are important for the study of induced recombination. Recombinogenic effects in yeast have long been used as a general indicator of genetic damage. The best characterized assays for recombinogens are those that detect mitotic crossing over and mitotic gene conversion in the yeast Saccharomyces cerevisiae.
Gene Mutations in Mammals
Mutagenicity assays in cultured mammalian cells have some of the same advantages as microbial assays with respect to speed and cost, and they follow quite similar approaches. The most widely used assays for gene mutations in mammalian cells detect forward mutations that confer resistance to a toxic chemical.
In vivo assays involve treating intact animals and analyzing genetic effects in appropriate tissues. Mutations may be detected either in somatic cells or in germ cells.
The mouse spot test is a traditional genetic assay for gene mutations in somatic cells. Visible spots of altered phenotype in mice heterozygous for coat color genes indicate mutations in the progenitor cells of the altered regions.
Mutation assays also provide information on mechanisms of mutagenesis. Base-pair substitutions and large deletions can be differentiated through the use of probes for the target gene and Southern blotting, in that base substitutions are too subtle to be detectable on the blots. Gene mutations have been characterized at the molecular level by DNA-sequence analysis both in transgenic rodents and in endogenous mammalian genes.
Transgenic animals are products of DNA technology in which the animal contains foreign DNA sequences that have been added to the genome and are transmitted through the germ line. The foreign DNA is therefore represented in all the somatic cells of the animal.
Mice that carry lac genes from E. coli use either lacI or lacZ as a target for mutagenesis. After mutagenic treatment of the transgenic animals, the lac genes are recovered from the animal, packaged into phage λ, and transferred to E. coli for mutational analysis. Mutant plaques are identified on the basis of phenotype, and mutant frequencies can be calculated for different tissues of the treated animals.
Mammalian Cytogenetic Assays
Genetic assays without DNA sequencing are indirect, in that one observes a phenotype and reaches conclusions about genes. In contrast, cytogenetic assays use microscopy for direct observation of the effect of interest. In conventional cytogenetics, metaphase analysis is used to detect chromosomal anomalies. Cells should be treated during a sensitive period of the cell cycle (typically S), and aberrations should be analyzed at the first mitotic division after treatment. Examples of chromosome aberrations are shown in Figure 9–3.
Chromosome aberrations induced by x-rays in Chinese hamster ovary (CHO) cells.A. A chromatid deletion (
. A chromatid exchange called a triradial (
. A small interstitial deletion (
) that resulted from chromosome breakage. D
. A metaphase with more than one aberration: a centric ring plus an acentric fragment (
) and a dicentric chromosome plus an acentric fragment (→).
It is essential that sufficient cells be analyzed because a negative result in a small sample is equivocal and inconclusive. Results should be recorded for specific classes of aberrations, not just as an overall index of aberrations per cell.
In interpreting results on the induction of chromosome aberrations in cell cultures, questionable positive results have been found at highly cytotoxic doses, high osmolality, and pH extremes. Although excessively high doses may lead to artifactual positive responses, the failure to test to sufficiently high doses also undermines the utility of a test; therefore, testing should be conducted at an intermediate dose and extended to a dose at which some cytotoxicity is observed.
In vivo assays for chromosome aberrations involve treating intact animals and later collecting cells for cytogenetic analysis. The main advantage of in vivo assays is that they include mammalian metabolism, DNA repair, and pharmacodynamics. The target is a tissue from which large numbers of dividing cells are easily prepared for analysis such as bone marrow.
In interphase cell analysis by fluorescence in situ hybridization (FISH), a nucleic acid probe is hybridized to complementary sequences in chromosomal DNA. The probe is labeled with a fluorescent dye so that the chromosomal location to which it binds is visible by fluorescence microscopy; often, probes are used that cover the whole chromosome, called “chromosome painting.”
Chromosome painting facilitates cytogenetic analysis, because aberrations are easily detected by the number of fluorescent regions in a painted metaphase. FISH permits the scoring of stable aberrations, such as translocations and insertions, which are not readily detected in traditional metaphase analysis of unbanded chromosomes.
Micronuclei are membrane-bounded structures that contain chromosomal fragments, or sometimes whole chromosomes, that were not incorporated into a daughter nucleus at mitosis. Micronuclei usually represent acentric chromosomal fragments, and they are commonly used as simple indicators of chromosomal damage. Micronuclei in a binucleate human lymphocyte are shown in Figure 9–4.
Micronucleus in a human lymphocyte. The cytochalasin B method was used to inhibit cytokinesis that resulted in a binucleate nucleus. The micronucleus resulted from failure of an acentric chromosome fragment or a whole chromosome being included in a daughter nucleus following cell division. (Image courtesy of James Allen, Jill Barnes, and Barbara Collins.)
Sister Chromatid Exchange
SCE, in which apparently reciprocal segments have been exchanged between the two chromatids of a chromosome, is visible cytologically through differential staining of chromatids (Figure 9–5). SCE assays are general indicators of mutagen exposure, rather than measures of a mutagenic effect.
Sister chromatid exchanges (SCEs) in human lymphocytes.A. SCEs in untreated cell. B. SCEs in cell exposed to ethyl carbamate. The treatment results in a very large increase in the number of SCEs. (Image courtesy of James Allen and Barbara Collins.)
Assays for aneuploidy include chromosome counting, the detection of micronuclei that contain kinetochores, and the observation of abnormal spindles or spindle–chromosome associations in cells in which spindles and chromosomes have been differentially stained.
The presence of the spindle attachment region of a chromosome (kinetochore) in a micronucleus can indicate that it contains a whole chromosome. Aneuploidy may therefore be detected by means of antikinetochore antibodies with a fluorescent label or FISH with a probe for centromere-specific DNA. Frequencies of micronuclei ascribable to aneuploidy and to clastogenic effects may therefore be determined concurrently by tabulating micronuclei with and without kinetochores.
Mammalian germ cell assays provide the best basis for assessing risks to human germ cells. Mammalian assays permit the measurement of mutagenesis at different germ cell stages. Late stages of spermatogenesis are often found to be sensitive to mutagenesis, but spermatocytes, spermatids, and spermatozoa are transitory. Mutagenesis in stem cell spermatogonia and resting oocytes is of special interest in genetic risk assessment because of the persistence of these stages throughout reproductive life.
Knowledge of the induction of chromosome aberrations in germ cells is important for assessing risks to future generations. A germ cell micronucleus assay has been developed, in which chromosomal damage induced in meiosis is measured by observation of rodent spermatids. Aneuploidy originating in mammalian germ cells may be detected cytologically through chromosome counting for hyperploidy or genetically in the mouse sex-chromosome loss test.
Besides cytologic observation, indirect evidence for chromosome aberrations is obtained in the mouse heritable translocation assay, which measures reduced fertility in the offspring of treated males. This presumptive evidence of chromosomal rearrangements can be confirmed through cytogenetic analysis.
Dominant Lethal Mutations
The mouse or rat dominant lethal assay offers an extensive database on the induction of genetic damage in mammalian germ cells. Commonly, males are treated on an acute or subchronic basis with the agent of interest and then mated with virgin females. The females are killed and necropsied during pregnancy so that embryonic mortality, assumed to be due to chromosomal anomalies, may be characterized and quantified.
Development of Testing Strategies
Concern about adverse effects of mutation on human health, principally carcinogenesis and the induction of transmissible damage in germ cells, has provided the impetus to identify environmental mutagens. Genetic toxicology assays may be used to screen chemicals to detect mutagens and to obtain information on mutagenic mechanisms and dose–responses that contribute to an evaluation of hazards. Besides testing pure chemicals, environmental samples are tested because many mutagens exist in complex mixtures. The analysis of complex mixtures often requires a combination of mutagenicity assays and refined analytical methods.
The first indication that a chemical is a mutagen often lies in chemical structure. Potential electrophilic sites in a molecule are alerts to possible mutagenicity and carcinogenicity, because such sites confer reactivity with nucleophilic sites in DNA.
Assessment of a chemical's genotoxicity requires data from well-characterized genetic assays. Sensitivity refers to the proportion of carcinogens that are positive in the assay, whereas specificity is the proportion of noncarcinogens that are negative. Sensitivity and specificity both contribute to the predictive reliability of an assay.
Rather than trying to assemble batteries of complementary assays, it is prudent to emphasize mechanistic considerations in choosing assays. Such an approach makes a sensitive assay for gene mutations (e.g., the Ames assay) and an assay for clastogenic effects in mammals pivotal in the evaluation of genotoxicity. Beyond gene mutations, one should evaluate damage at the chromosomal level with a mammalian in vitro or in vivo cytogenetic assay. Other assays offer an extensive database on chemical mutagenesis (Drosophila SLRL), a unique genetic endpoint (i.e., aneuploidy; mitotic recombination), applicability to diverse organisms and tissues (i.e., DNA damage assays, such as the comet assay), or special importance in the assessment of genetic risk (i.e., germ cell assays).