In Vitro Genetic Toxicity Testing

1. In Vitro Genetic Toxicity Testing Dr M. Moshiri

2. In vitro genetic toxicity tests, concerned with the genetic effects of toxic materials in vitro, are the forerunner of all in vitro toxicity testing. • In contrast to in vitro tests measuring only toxic effects, but often in cellular systems selected to mimic target tissue and organ toxicity, genotoxicity tests are selected for their precision in assessing genetic effects in surviving cells

3. About 5,000 diseases in humans are now known to be caused by defective genes. • These inherited disorders cause 20% of all infant mortalities • Half (50%) of all miscarriages • 80% of all cases of mental retardation.

4. BASIC MECHANISMS • Genetic toxicology test systems measure the outcome of damage or alterations in DNA • If DNA is damaged, it may : • be correctly repaired with no genetic consequences • lead to cell death, again with no genetic consequences • be replicated with the damage incorrectly repaired. • Only the third consequence leads to mutations, DNA alterations propagated through subsequent generations of cells or individuals.

5. BASIC MECHANISMS • Genetic toxicology tests assessingDNA damage and repair, such as SCE tests and tests for UDS (the repair of DNA damage at times other than the scheduled phase of the mitotic cycle, i.e., the S-phase), are less frequently used for regulatory submissions today because they provide only indirect evidence of mutagenesis. • Instead, the harmonized testing approaches for regulatory submissions, which will be described, consist of tests for gene and chromosomal mutations.

6. Gene Mutations • Gene mutations can be assessed in bacteria, mammalian cells in culture, and whole organisms

7. Gene Mutations Gene mutations in bacteria • A base-pair substitution mutation =a single nucleotide is changed a subsequent change in the complementary nucleotide on the other strand of the DNA double helix. • Such mutations are deleterious when they alter a protein coding sequence to • Conclude translation prematurely (a nonsense mutation) • incorporate a different amino acid (a missense mutation).

8. Gene Mutations Gene mutations in bacteria • Frameshift mutations occur after the deletion or insertion of one nucleotide, which then changes the “reading frame” for the remainder of the gene, or even for multiple genes.

9. Gene Mutations Gene mutations in bacteria • Both base-pair substitution and frameshift mutations are routinely measured in bacterial cells by • the cells’ acquisition of the capability of growth in an environment containing a missing amino acid • For these tests, a large number of bacteria is examined to demonstrate significant increases over spontaneous mutation frequencies. • A forward mutationoccurs when a change takes place in the native DNA • A reverse mutationoccurs when a mutated cell is returned to its initial phenotype. • The currently used bacterial tests are reverse mutation assays.

10. Gene Mutations Gene mutations in mammalian cells. • Generally forward mutations • Include base-pair substitution and frameshift mutations. • Measurements of gene mutations in mammalian cells reflect the greater complexity of mammalian cells and chromosomes in comparison to those of prokaryotes, and, thus, they more closely approximate the genetic effects of chemicals in rodent species and humans.

11. Gene Mutations Gene mutations in mammalian cells. • In contrast to bacteria, mammalian cells are essentially diploid (2n, with two copies of each chromosome). • Mammalian chromosomes contain nonfunctional ( noncoding), as well as functional(coding) sequences. • Usually two copies of each gene exist (one on each chromosome) • One (dominant) form of the gene may be expressed while the other (recessive) gene remains unexpressed, unless both copies are recessive.

12. Gene Mutations Gene mutations in mammalian cells. • Homozygous = both copies of the gene are the same • Heterozygous= the copies are different • Hemizygous = only one chromosome is present to carry the trait. • Hemizygous traits are found on the X chromosome in mammals because males have only one X chromosome, and only one X chromosome is expressed in female cells.

13. Gene Mutations Gene mutations in vivo • In spite of the early promise of transgenic mutagenesis systems, they have been found to lack sensitivity and to be capable of detecting only a few of the chemicals known to be rodent carcinogens. • Therefore, such tests are currently identified as optional, but not required, systems for regulatory submissions, and no universally accepted and routinely used test system exists for assessing gene mutations in vivo.

14. Chromosomal Mutations • Chromosomal mutations are large-scale numerical or structural alterations in eukaryotic chromosomes that may affect the expression of numerous genes with gross effects, or be lethal to affected cells . • Including • Small and large deletions (visualized as breaks) • Translocations (exchanges) • Nondisjunction (aneuploidy) • Mitotic recombination • Chromosomal abnormalities are associated with neoplasia,spontaneous abortion, congenital malformation, and infertility, which occur in approximately 0.6% of live births in humans. • It has been estimated that up to 40% of spontaneous abortuseshave chromosomal defects and essentially all tumors harbor chromosomal mutations.

15. Chromosomal Mutations • Chromosomal mutations in mammalian cells. • The L5178Ymouse lymphoma assay is routinely used • Because it is the most extensively characterized of the several assays.

16. Chromosomal Mutations • Chromosomal mutations in mammalian cells. • L5178Ymouse lymphoma assay • the L5178YTK+/- mouse lymphoma-TK assay detects the mutations at the thymidinekinase locus caused by basepair changes, frameshift and small deletions. • Mutant cells, deficient in TK due to the forward mutation in the TK locus (from TK+ to TK-), are resistant to the cytotoxic effect of pyrimidine analogues such as trifluorothymidine (TFT). • The mutagenicity of the test agents is indicated by the increase in the number of mutants after treatment.

17. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo • In contrast to the described assays, which assess gene and chromosomal mutations at only one or a few genes, in millions of cells per treatment, the in vitro and in vivo assays for chromosomal aberrations assess mutagenic events in multiple genes, but usually for no more than 200 cells per culture, or for up to 100 cells per animal.

18. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • Chromosome breakage, necessary for chromosomal rearrangements, is the classic endpoint in chromosomal aberration assays. • To visualize chromosomes and chromosomal aberrations with a light microscope • after in vitro or in vivo treatment with a chemical • cells are arrested in metaphase • treated with a hypotonic solution to swell the chromosomes • fixed ,transferred to microscope slides, and stained

19. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • The first metaphase (M) after chemical exposure, M1, is the time when the greatest number of chromosomally damaged cells may be observed, • the extent of damage declines rapidly after M1 intherphse prophse Early telophse Late telophse Metaphse Anaphse

20. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • When the chromosomes of diploid somatic cells are replicated, each chromosome then consists of two (sister) chromatids separating at mitosis to become the chromosomes of the daughter cells. • If chromosomal mutations occur before replication (DNA synthesis), both chromatids will be affected. • This damage will be visualized as chromosomal breaks (deletions) and exchanges(translocations).

21. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • If these mutations occur during replication (the most sensitive stage), or after replication, the damage is visualized as chromatidbreaks and exchanges.

22. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • Hence, by enumerating chromatid and chromosome breaks and exchanges, an index can be obtained of the time that the damage occurred. • Very large deletions are tolerated only if they do not incapacitate essential genes.

23. Chromosomal Mutations • Chromosomal aberrations • In vitro and in vivo. • Although not currently used in cytogenetic testing for regulatory submissions, fluorescence in situ hybridization (FISH) staining techniques have been recently developed for human and mouse chromosomes, in which each chromosome can be differentially stained, revealing chromosomalrearrangements not apparent with conventional staining techniques • When FISH staining is translated from a research approach to a testing protocol, it may be possible to reduce the number of chromosomes to be analyzed and, hence, the time for chromosomal aberration tests.

24. Chromosomal Mutations Micronuclei: Micronuclei result when nuclear membranes form around broken pieces of chromosomes or around chromosomes failing to separate at cell division. • Therefore, micronucleus tests measure • chromosome breakage, the classic endpoint for chromosomal aberration assays • aneuploidy, the loss or gain of a chromosome or a chromosome segment.

25. Chromosomal Mutations • Micronuclei: • In vitro micronucleus tests are currently under development in a number of laboratories as a less subjective and more economical alternative to in vitro chromosomal aberration tests. • For these approaches, cytochalasin B is used to arrest cell division (cytokinesis) but not nuclear division, and up to 1,000 binucleate cells are examined for the presence or absence of micronuclei. • because the in vitro micronucleus tests have yet to be validated and shown to be at least as effective as tests for chromosomal aberrations in vitro, none is currently recommended for regulatory Submissions.

26. Chromosomal Mutations • Micronuclei: • In vivo micronucleus tests are justified for regulatory submissions for assessing chromosomal breakage and aneuploidy in an environment including in vitro metabolic reactions. • Micronuclei are readily observed microscopically in stained preparations of (otherwise anucleate) polychromatic erythrocytes (PCEs) from the bone marrow of rats or mice or from the peripheral blood of mice; the latter because, in mice, the spleen does not remove micronucleated cells from the blood. • With appropriate staining techniques, the PCEs can be differentiated from the more mature normochromatic erythrocytes (NCEs) because the PCEs still contain RNA, which has been lost by the NCEs. • For example, with Giemsa staining, the PCEs are blue and the NCEs are salmon pink or red.

27. Chromosomal Mutations • Micronuclei: • Peripheral blood erythrocytes can be obtained for micronucleus evaluations • without sacrificing the animal, • a greater number of cells must be evaluated because the newly formed erythrocytes (PCEs, the cells of interest) are diluted in the population of preexisting erythrocytes. • Bone marrow cells • give amore informative index of toxicity • are routinely used for the micronucleus test.

28. GENETIC TOXICOLOGY TESTS FOR CURRENT PRODUCT REGISTRATION • Current OECD SIDS and ICH regulatory guidance has identified the following five basic genetic toxicology approaches • A test for bacterial reverse gene mutations • the L5178Y mouse lymphoma cell assay for gene and chromosomal mutations • An in vitro chromosomal aberration test, and either • An in vivo chromosomal aberration test or • An in vivo micronucleus test, • TSCA (Toxic Substances Control Act) regulatory guidance has identified all but the in vitro chromosomal aberration test.

29. GENETIC TOXICOLOGY TESTS FOR CURRENT PRODUCT REGISTRATION • To establish that a chemical is negative • in vitro tests must be conducted in the absence and presence of exogenous metabolic activation • Positive and negative controls must be within historical ranges for the testing laboratory • Testing must be conducted to a level at which toxicity or precipitation is observed or to a level for which higher concentrations would not yield biologically relevant results. • The latter two requirements are also applicable for in vivo tests.

30. GENETIC TOXICOLOGY TESTS FOR CURRENT PRODUCT REGISTRATION • In addition, the animals must be maintained under conditions minimizing the influence of environmental variables, and bioavailability must be considered when selecting the route of administration. • It should also be noted that, although some prior testing guidelines specified that test results should be reproducible in independent experiments, under current guidelines, no requirement exists for repeating an appropriately conducted test yielding clearly positive results.

31. Reverse Gene Mutations in Bacteria • The most extensive testing for gene mutations is in bacteria, particularly using reverse mutation in Salmonella typhimurium and Escberichia coli. • Therefore, bacterial reverse mutation assays are considered by many researchers to be the cornerstone of genetic toxicology testing. • Advantages • relative ease of performance • Economy • Efficiency • the ability to identify specific DNA damage that is induced, e.g., frameshift or base-pair substitution mutations • Bacterial tests can also provide information on the mode of action of the test chemical, because the bacterial strains used vary in their responsiveness to different chemical classes. • Many of the tester strains have features making them more sensitive for the detection of mutations, including • responsive DNA sequences at the reversion sites • increased cell permeability to large molecules • the elimination of DNA repair systems or the enhancement of error-prone DNA repair processes.

32. Reverse Gene Mutations in Bacteria • The S. typhimuriumstrains routinely used were designed for sensitivity in detecting gene mutations reverting the bacteria to histidine independence • the Salmonella strains are histidineauxotrophsby virtue of mutations in the histidineoperon. • The E. coli WP2 uvrA strains recommended for initial tests are tryptophan auxotrophsby virtue of a base-pair substitution mutationin the tryptophan operon. • When these histidine- or tryptophan-dependent cells are grown on minimal medium agar plates containing a trace of histidine or tryptophan, only those cells reverting to histidine or tryptophan independence are able to form colonies.

33. Reverse Gene Mutations in Bacteria • In addition to the histidine and tryptophan operons, most of the indicator strains carry a deletion covering genes involved in the synthesis of the vitamin biotin (bio), and all carry the rfa mutation leading to a defective lipopolysaccharide coat and making the strains more permeable to many large molecules. • The strains also carry the uvrB mutation, which results in impaired repair of ultraviolet (UV)-induced DNA damage and renders the bacteria unable to use accurate excision repair to remove certain chemically or physically damaged DNA, thereby enhancing the strains’ sensitivity to some mutagenic agents.

34. Reverse Gene Mutations in Bacteria • In bacterial reverse mutation testing, usually one strain is used for a preliminary concentration range-finding assay, and then mutagenesis assays are conducted with five strains. The strains recommended by the OECD guidelines are: • S. typhimurium TA1535 • S. typhimurium TA1537 or TA97 or TA97a • S. typhimurium TA98 • S. typhimurium TA100 (histidine independence by base-pair mutagens) • E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101) or S. typhimurium TA102

35. Reverse Gene Mutations in Bacteria • In the standard plate incorporation protocol • the test material, bacteria, and either a metabolic activation mixture [9, 000-g postmitochondrial supernatant (S9)] or a buffer are added to liquid top agar • Agar in a disposable glass tube, which is held at 45°C in a heating block while the components are added, then mixed, and the mixture is immediately poured on a plate of bottom agar. • After the agar gels, the bacteria are incubated, at 37°C, for 48–72 hours; then, the resulting colonies are counted. • In a typical mutagenesis assay with and without metabolic activation, each of the five strains of bacteria is exposed to the solvent control (with six cultures per strain and activation condition), and to five concentrations of the test chemical (with three cultures per concentration, strain, and activation condition), and to the appropriate positive controls for that strain (with three cultures per activation condition). • This process yields a total of 240 bacterial plates and additional plates used to check the sterility of the components.

36. Reverse Gene Mutations in Bacteria • The preincubation modification • this method is used for materials that may be poorly detected in the plate incorporation assay, including • short chain aliphatic nitrosamines • divalent metals • aldehydes • azo-dyes and diazo compounds • pyrollizidine alkaloids • alkyl compounds • nitro compounds. • In this protocol, the test material, bacteria, and S9 mixture (when used) are incubated for 20–30 minutes at 37°C before top agar is added, mixed, and the mixture is poured on a plate of bottom agar.

37. Reverse Gene Mutations in Bacteria • For an acceptable assay, the test chemical should be tested to a toxic level, • as evidenced by a reduction in colonies or a reduced background lawn • to a level at which precipitated test material precludes visualization of the colonies or • to 5 mg or 5 μl/plate whichever is lower. • To evaluate a result as positive requires • a concentration-related Or • a reproducible increase in the number of revertant colonies per plate for at least one strain with or without activation.

38. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • The L5178Y mouse lymphoma assay measures gene and chromosomal forward mutations at the tk locus, tk+/−−−>tk−/−/− • As indicated in the recent EPA Gene-Toxreview of published results for over 600 chemicals tested in this assay , • When used with appropriate protocols and evaluation criteria, the mouse lymphoma assay yields results at least 95% concordant with the outcome of the rodent carcinogenesis bioassay. http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPackaging/Redbook/ucm078336.htm

39. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • More recently, the L5178Y mouse lymphoma cells were found to harbor gene mutations p53 ( in the mouse, is found on the same chromosome as tk.) • The p53 tumor suppressor gene is considered to be the “guardian of the genome” because its function is to delay the cell-cycle progression of cells that have acquired chromosomal mutations until the damage has been repaired. • Thus, the presence of mutant p53 in the mouse lymphoma cells renders the assay more similar to mutation assays in repair-deficient bacteria. • this finding is not only consistent with the sensitivity of this assay for detecting chromosomal mutations, but it enhances the relevance of the assay for predicting carcinogenicity, as mutant p53 is found in over 50% of human tumors.

40. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • L5178Y mouse lymphoma cells grow in suspension culture with a relatively short cell generation time, 9–10 hours. • A few days before use in an assay, a culture is “cleansed” of preexisting spontaneous tk−/− mutants by growing the cells for about 24 hours in medium containing methotrexate. • After the cells have recovered from cleansing, they are exposed to a series of concentrations of the test chemical, usually for 4 hours, in the absence and presence of metabolic activation. • Testing under nonphysiologic conditions must be avoided in this assay and in other in vitro mammalian cell assays, as acidic pH shifts, to ≤6.5, and high salt concentrations have been shown to produce physiologically irrelevant positive results. • Conversely, if the pH of the medium used to culture the cells is ≥7.5, cell growth in suspension culture may be depressed, and small colony mutants, in particular, may not be detected

41. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • Because chromosomal mutations are usually associated with slower growth rates and because the induction of both gene and chromosomal mutations are associated with cytotoxicity, • A chemical cannot be considered to be nongenotoxic in this assay unless testing is performed to concentrations producing significant cytotoxicity,

42. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • Cytotoxicity should be determined for each individual test and control culture. • For the soft agar version of the MLA, this has generally been done using the relative total growth (RTG) • This measure includes the relative growth in suspension during the expression time and the relative cloning efficiency at the time that mutants are selected. • The microwell version of the assay was developed using the relative survival (RS) as the cytotoxicity measure. • The RS is determined by the relative plating efficiency of each culture when plated immediately after the exposure period • The RTG and the RS are different measures of cytotoxicity and, although there is no real justification that one measure is superior to the other, it is important that the same measure of cytotoxicity be used for both versions of the assay. • Because the RS is not normally measured in the soft agar version of the assay and the RTG is measured in both versions, it is recommended that the RTG be used as the standard measure of cytotoxicity.

43. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • On the other hand, responses observed only at extreme cytoxicity (<10% RTG) are considered to be biologically irrelevant. • Therefore, exposure concentrations for each assay are selected, based on the results of a preliminary range-finding experiment, • to span a range of anticipated survival from nontoxic or weakly toxic to 10–20% RTG, with the concentrations selected to emphasize the lower RTG values. • For relatively noncytotoxic chemicals, the maximum concentration should be • 5 μl/ ml, • 5 mg/ml, • 10 mM, • A concentration evidencing insolubility whichever is lower.

44. The L5178Y/ThymidineKinase+/− Gene and Chromosomal Mutation Assay • Procedure 1. Treatment with test substance • Cells, growing in log phase, should be exposed to the test substance both with and without metabolic activation. Exposure should be for a suitable period of time (generally 3-4 hrs is used). 2.Expression time and measurement of mutant frequency • At the end of the exposure period, cells are washed and cultured to allow for the expression of the mutant phenotype (in suspension culture for a 2-day expression period) • the cells are cloned to measure mutagenesis and survival • Two methods are currently used for cloning : • cloned in culture dishes in a medium containing sufficient soft agar to immobilize the cells (2 WEEK) • cloning the cells without agar in microwell plates(10-12 days) 3. Mutant Colony Sizing

46. Chromosomal Aberrations: In Vitro • In vitro chromosomal aberration assays for regulatory submissions are routinely conducted in the absence and presence of exogenous metabolic activation and may use • a variety of established cell lines • cell strains, • primary cell cultures • selected on the basis of factors such as • growth ability in culture • stability of the karyotype • chromosome number • chromosome diversity • spontaneous frequency of chromosome aberrations. • The most frequently used cells are Chinese hamster fibroblasts (either CHO or Chinese hamster lung (CHL) cells) or human or rat lymphocytes stimulated to divide synchronously in vitro. • Established cell cultures present the advantages of minimal variability among experiments

47. Chromosomal Aberrations: In Vitro • The cells are propagated from stock cultures and seeded in a culture medium at a density such that the cultures will not reach confluency before the time of harvest, which should be ~1.5 hours after the addition of a mitotic spindle inhibitor (Colcemid or colchicine). • Human lymphocyte cultures are used because of • their perceived relevance to the human condition • to presenting the advantage of synchronous cell division. • Variability in mitogenicresponse can be minimized by using cultures of rat lymphocytes.

48. Chromosomal Aberrations: In Vitro • The lymphocytes are usually obtained from whole blood from healthy (human or rodent) subjects, treated with heparin (an anticoagulant), and stimulated to divide with a mitogen(e.g., phytohemagglutinin). • After a prolonged G1 stage, the cells enter S-phase, which should be the time of addition of the test chemical to the cells. • chemical exposure is initiated at 48 hours • a mitotic spindle inhibitor is added at 70.5 hours • cells in metaphase are harvested at 72 hours. • After the cells are harvested, they are treated with a hypotonic solution to swell the chromosomes  fixed,  dropped onto prelabeled slides  The chromosomes are stained  cover slips are attached to permit microscopic analysis with oil immersion (100X) objectives.

49. Chromosomal Aberrations: In Vitro At least three concentrations are analyzed that are selected based on a preliminary evaluation of uncoded slides.

50. Chromosomal Aberrations: In Vitro • Among the criteria to be considered when determining the highest concentration to be tested for chromosomal aberrations are • cytotoxicity • Solubility • and changes in osmolality or pH to ensure that exposure conditions will be in a physiologically relevant range • As a general rule-of-thumb, to ensure that • a sufficient number of mitotic cells for analyses exist • mitotic indices need not be depressed more than 50%. • For relatively noncytotoxic chemicals, the maximum concentration should be • 5μl/ml • 5 mg/ml • 10 mM, whichever is lowest. • For relatively insoluble chemicals, OECD guidelines advise • testing one or more than one concentration in the insoluble range as long as a precipitate does not interfere with the analysis.