API

Event: 185

Key Event Title

?

Increase, Mutations

Short name

?

Increase, Mutations

Biological Context

?

Level of Biological Organization
Molecular

Cell term

?


Organ term

?


Key Event Components

?

Process Object Action
mutation deoxyribonucleic acid increased

Key Event Overview


AOPs Including This Key Event

?


Stressors

?


Taxonomic Applicability

?

Term Scientific Term Evidence Link
Mus musculus Mus musculus High NCBI
medaka Oryzias latipes Moderate NCBI
rat Rattus norvegicus High NCBI
Homo sapiens Homo sapiens Moderate NCBI

Life Stages

?

Life stage Evidence
All life stages High

Sex Applicability

?

Term Evidence
Unspecific High

Key Event Description

?


A mutation is a change in DNA sequence. Mutations can thus alter the coding sequence of genes, potentially leading to malformed or truncated proteins. Mutations can also occur in promoter regions, splice junctions, non-coding RNA, DNA segments, and other functional locations in the genome. These mutations can lead to various downstream consequences, including alterations in gene expression. There are several different types of mutations including missense, nonsense, insertion, deletion, duplication, and frameshift mutations, all of which can impact the genome and its expression in unique ways.

 

Mutations can be propagated to daughter cells upon cellular replication. Mutations in stem cells (versus terminally differentiated non-replicating cells) are the most concerning, as these will persist in the organism. The consequence of the mutation, and thus the fate of the cell, depends on the location (e.g., coding versus non-coding) and the type (e.g., nonsense versus silent) of mutation.

Mutations can occur in somatic cells or germ cells (sperm or egg).


How It Is Measured or Detected

?


Mutations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Some examples are given below.

Somatic cells: The Salmonella mutagenicity test (Ames Test) is generally used as part of a first tier screen to determine if a chemical can cause gene mutations. This well-established test has an OECD test guideline (TG 471). A variety of bacterial strains are used, in the presence and absence of a metabolic activation system (e.g., rat liver microsomal S9 fraction), to determine the mutagenic potency of chemicals by dose-response analysis. A full description is found in Test No. 471: Bacterial Reverse Mutation Test (OECD).

A variety of in vitro mammalian cell gene mutation tests are described in OECD’s Test Guidelines 476 and 490. TG 476 is used to identify substances that induce gene mutations at the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene, or the transgenic xprt (xanthine-guanine phosphoribosyl transferase) reporter locus. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. The only cells suitable for the XPRT test are AS52 cells containing the bacterial xprt (or gpt) transgene (from which the hprt gene was deleted).

The new OECD TG 490 describes two distinct in vitro mammalian gene mutation assays using the thymidine kinase (tk) locus and requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. The autosomal and heterozygous nature of the thymidine kinase gene in the two cell lines enables the detection of cells deficient in the enzyme thymidine kinase following mutation from tk+/- to tk-/-.

It is important to consider that different mutation spectra are detected by the different mutation endpoints assessed. The non-autosomal location of the hprt gene (X-chromosome) means that the types of mutations detected in this assay are point mutations, including base pair substitutions and frameshift mutations resulting from small insertions and deletions. Whereas, the autosomal location of the transgenic xprt, tk, or gpt locus allows the detection of large deletions not readily detected at the hemizygous hprt locus on X-chromosomes. Genetic events detected using the tk locus include both gene mutations (point mutations, frameshift mutations, small deletions) and large deletions.

The transgenic rodent mutation assay (OECD TG 488) is the only assay capable of measuring gene mutation in virtually all tissues in vivo. Specific details on the rodent transgenic mutation reporter assays are reviewed in Lambert et al. (2005, 2009). The transgenic reporter genes are used for detection of gene mutations and/or chromosomal deletions and rearrangements resulting in DNA size changes (the latter specifically in the lacZ plasmid and Spi- test models) induced in vivo by test substances (OECD, 2009, OECD, 2011; Lambert et al., 2005). Briefly, transgenic rodents (mouse or rat) are exposed to the chemical agent sub-chronically. Following a manifestation period, genomic DNA is extracted from tissues, transgenes are rescued from genomic DNA, and transfected into bacteria where the mutant frequency is measured using specific selection systems.

The Pig-a (phosphatidylinositol glycan, Class A) gene on the X chromosome codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Cells lacking GPI anchors, or GPI-anchored cell surface proteins are predominantly due to mutations in the Pig-a gene. Thus, flow cytometry of red blood cells expressing or not expressing the Pig-a gene has been developed for mutation analysis in blood cells from humans, rats, mice, and monkeys. The assay is described in detail in Dobrovolsky et al. (2010). Development of an OECD guideline for the Pig-a assay is underway. In addition, experiments determining precisely what proportion of cells expressing the Pig-a mutant phenotype have mutations in the Pig-a gene are in progress (e.g., Nicklas et al., 2015, Drobovolsky et al., 2015). A recent paper indicates that the majority of CD48 deficient cells from 7,12-dimethylbenz[a]anthracene-treated rats (78%) are indeed due to mutation in Pig-a (Drobovolsky et al., 2015).


Germ cells: Tandem repeat mutations can be measured in bone marrow, sperm, and other tissues using single-molecule PCR. This approach has been applied most frequently to measure repeat mutations occurring in sperm DNA. Isolation of sperm DNA is as described above for the transgenic rodent mutation assay, and analysis of tandem repeats is done using electrophoresis for size analysis of allele length using single-molecule PCR. For expanded simple tandem repeat this involved agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al. (2002) and Beal et al. (2015).

Mutations in rodent sperm can also be measured using the transgenic reporter model (OECD TG 488). A description of the approach is found within this published TG. Further modifications to this protocol have now been made for the analysis of germ cells. Detailed methodology for detecting mutant frequency arising in spermatogonia is described in Douglas et al. (1995), O'Brien et al. (2013); and O'Brien et al. (2014). Briefly, male mice are exposed to the mutagen and killed at varying times post-exposure to evaluate effects on different phases of spermatogenesis. Sperm are collected from the vas deferens or caudal epididymis (the latter preferred). Modified protocols have been developed for extraction of DNA from sperm.

A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).


Please note, gene mutations that occur in somatic cells in vivo (OECD Test. No. 488) or in vitro (OECD Test No. 476: In vitro Mammalian Cell Gene Mutation Test), or in bacterial cells (i.e., OECD Test No. 471) can be used as an indicator that mutations in male pre-meiotic germ cells may occur for a particular agent (sensitivity and specificity of other assays for male germ cell effects is given in Waters et al., 1994). However, given the very unique biological features of spermatogenesis relative to other cell types, known exceptions to this rule, and the small database on which this is based, inferring results from somatic cell or bacterial tests to male pre-meiotic germ cells must be done with caution. That mutational assays in somatic cells may predict mutations in germ cells has not been rigorously tested empirically (Singer and Yauk, 2010). The IWGT working group on germ cells specifically addressed this gap in knowledge in their report (Yauk et al., 2015) and recommended that additional research address this issue. Mutations can be directly measured in humans (and other species) through the application of next-generation sequencing. Although single-molecule approaches are growing in prevalence, the most robust approach to measure mutation using next-generation sequencing today requires clonal expansion of the mutation to a sizable proportion (e.g., sequencing tumours; Shen et al., 2015), or analysis of families to identify germline derived mutations (reviewed in Campbell and Eichler, 2013; Adewoye et al., 2015).

Please refer to the table below for additional details and methodologies for measuring mutations.

Assay Name References Description OECD Approved Assay
Assorted Gene Loci Mutation Assays

Tindall et al., 1989; Kruger et al., 2015

After exposure to a chemical/mutagen, mutations can be measured by the ability of exposed cells to form colonies in the presence of specific compounds that would normally inhibit colony growth; Usually only cells -/- for the gene of interest are able to form colonies N/A
TK Mutation Assay

Yamamoto et al., 2017; Liber et al., 1982; Lloyd and Kidd, 2012

After exposure to a chemical/mutagen, mutations are detected at the thymidine kinase (TK) loci of L5178Y wild-type mouse lymphoma TK (+/-) cells by measuring resistance to lethaltriflurothymidine (TFT); Only TK-/- cells are able to form colonies Yes (No. 490)
HPRT Mutation Assay

Ayres et al., 2006; Parry and Parry, 2012

Similar to TK Mutation Assay above, X-linked HPRT mutations produced in response to chemical/mutagen exposure can be measured through colony formation in the presence of 6-TG or 8-azoguanine; Only HPRT-/- cells are able to form colonies Yes (No. 476)
Salmonella Mutagenicity Test (Ames Test) OECD, 1997 After exposure to a chemical/mutagen, point mutations are detected by analyzing the growth capacity of different bacterial strains in the presence and absence of various metabolic activation systems  Yes (No. 471)
PIG-A / PIG-O Assay

Kruger et al., 2015; Nakamura, 2012; Chikura, 2019

After exposure to a chemical/mutagen, mutations  in PIG-A or PIG-O (which decrease the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor protein) are assessed by the colony-forming capabilities of cells after in vitro exposure, or by flow cytometry of blood samples after in vivo exposure N/A
Single Molecule PCR

Kraytsberg, 2005; Yauk, 2002

This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies N/A
ACB-PCR

Myers et al., 2014 (Textbook, pg 345-363); Banda et al.,  2013; Banda et al.,  2015; Parsons et al., 2017

Using this PCR technique, single base pair substitution mutations within oncogenes or tumour suppressor genes can be detected by selectively amplifying specific point mutations within an allele and selectively blocking amplification of the wild-type allele N/A
Transgenic Rodent Mutation Assay

OECD 2013; Lambert 2005; Lambert 2009

This in vivo test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After in vivo exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype Yes (No. 488)
Conditionally inducible transgenic mouse models Parsons 2018 (Review) Inducible mutations linked to fluorescent tags are introduced into transgenic mice; Upon exposure of the transgenic mice to an inducing agent, the presence and functional assessment of the mutations can be easily ascertained due to expression of the linked fluorescent tags N/A
Error-Corrected Next Generation Sequencing (NGS) Salk 2018 (Review) This technique detects rare subclonal mutations within a pool of heterogeneous DNA samples through the application of new error-correction strategies to NGS; At present, few laboratories in the world are capable of doing this, but commercial services are becoming available (e.g., Duplex sequencing at TwinStrand BioSciences) N/A

 


Domain of Applicability

?


Mutations can occur in any organism and in any cell type, and are the fundamental material of evolution. The test guidelines described above range from analysis from prokaryotes, to rodents, to human cells in vitro. Mutations have been measured in virtually every human tissue sampled in vivo.


Evidence for Perturbation by Stressor



Regulatory Significance of the Adverse Outcome

?



References

?


Adewoye, A.B., Lindsay, S.J., Dubrova, Y.E. and M.E. Hurles (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", Nat. Commun., 6:6684.

Ayres, M. F., da Cruz, D. A., Steele, P., & Glickman, W. B. 2006. Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway. Genetics and Molecular Biology. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.

Banda M, Recio L, Parsons BL. 2013. ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver. Environ Mol Mutagen. 54(8):659-67. Doi:10.1002/em.21808.

Banda M, McKim KL, Haber LT, MacGregor JA, Gollapudi BB, Parsons BL. 2015. Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation. Mutat Res Genet Toxicol Environ Mutagen. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003

Campbell, C.D. and E.E. Eichler (2013), "Properties and rates of germline mutations in humans", Trends Genet., 29(10): 575-84.

Chikura S, Kimoto T, Itoh S, Sanada H, Muto S, Horibata K. 2019. Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society. Genes Environ.  27:41-5. Doi: 10.1186/s41021-019-0121-z.

Dobrovolsky, V.N., J. Revollo, M.G. Pearce, M.M. Pacheco-Martinez and H. Lin (2015), "CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene. CD48-Deficient T-Lymphocytes from DMBA-Treated Rats Have De Novo Mutations in the Endogenous Pig-a Gene", Environ. Mol. Mutagen., 6(: 674-683.

Douglas, G.R., J. Jiao, J.D. Gingerich, J.A. Gossen and L.M. Soper (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", Proceedings of the National Academy of Sciences of the United States of America, 92(16): 7485-7489.

Kraytsberg Y, Khrapko K. 2005. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations. Expert Rev Mol Diagn. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.

Krüger, T. C., Hofmann, M., & Hartwig, A. 2015. The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells. Arch Toxicol. 89(12), 2429-43. doi: 10.1007/s00204-014-1413-5.

Lambert, I.B., T.M. Singer, S.E. Boucher and G.R. Douglas (2005), "Detailed review of transgenic rodent mutation assays", Mutat Res., 590(1-3):1-280.

Liber, L. H., & Thilly, G. W. 1982. Mutation assay at the thymidine kinase locus in diploid human lymphoblasts. Mutation Research. 94: 467-485. Doi:10.1016/0027-5107(82)90308-6.

Lloyd, M., & Kidd, D. 2012. The Mouse Lymphoma Assay. In: Parry J., Parry E. (eds) Genetic Toxicology. Methods in Molecular Biology (Methods and Protocols), 817. Springer, New York, NY.

Myers, M. B. et al., (2014), ACB-PCR Quantification of Somatic Oncomutation. Molecular Toxicology Protocols, Methods in Molecular Biology. DOI: 10.1007/978-1-62703-739-6_27

Nakamura, J. et al., (2012), Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system. Doi:10.1371/journal.pone.0033563.

Nicklas, J.A., E.W. Carter and R.J. Albertini (2015), "Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line", Environ. Mol. Mutagen., 6(8):663-73.

Norris, M.B. and R.N. Winn (2010), "Isolated spermatozoa as indicators of mutations transmitted to progeny", Mutat Res., 688(1-2): 36–40.

O'Brien, J.M., A. Williams, J. Gingerich, G.R. Douglas, F. Marchetti and C.L. Yauk (2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", Mutat. Res., 741-742:11-7.

O'Brien, J.M., M.A. Beal, J.D. Gingerich, L. Soper, G.R. Douglas, C.L. Yauk and F. Marchetti (2014), "Transgenic rodent assay for quanitifying male germ cell mutation frequency", Journal of Visual Experimentation, Aug 6;(90).

O’Brien, J.M., M. Walker, A. Sivathayalan, G.R. Douglas, C.L. Yauk and F. Marchetti (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", Environ. Mol. Mutagen., 6(4): 347-355.

OECD (1997), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (1997), Test No. 476: In vitro Mammalian Cell Gene Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2009), Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, N° 103, ENV/JM/MONO 7, OECD, Paris.

OECD (2011), Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2015), Test. No. 490: In vitro mammalian cell gene mutation mutation tests using the thymidine kinase gene, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD. 2013. Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays.

OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD. 2015. Test. No. 490: In vitro mammalian cell gene mutation mutation tests using the thymidine kinase gene. OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

Parry MJ, & Parry ME. 2012. Genetic Toxicology Principles and Methods. Humana Press. Springer Protocols.

Parsons BL, McKim KL, Myers MB. 2017. Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas. Environ Mol Mutagen. 58(7):466-476. Doi: 10.1002/em.22110.

Parsons BL. Multiclonal tumor origin: Evidence and implications. Mutat Res. 2018. 777:1-18. doi: 10.1016/j.mrrev.2018.05.001. Shen, T., S.H. Pajaro-Van de Stadt, N.C.

Salk JJ, Schmitt MW, Loeb LA. Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations. Nat Rev Genet. 2018. 19(5):269-285. doi: 10.1038/nrg.2017.117.

Shen, T., S.H. Pajaro-Van de Stadt, N.C. Yeat and J.C. Lin (2015), "Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes" Front. Genet., 6: 215. .

Singer, T.M. and C.L. Yauk CL (2010), "Germ cell mutagens: risk assessment challenges in the 21st century", Environ. Mol. Mutagen., 51(8-9): 919-928.

Tindall, R. K., & Stankowski Jr., F. L. 1989. Molecular analysis of spontaneous mutations at the GPT locus in Chinese hamster ovary (AS52) cells. Mutation Research, 220, 241-53. Doi: 10.1016/0165-1110(89)90028-6.

Waters, M.D., H.F. Stack, M.A. Jackson, B.A. Bridges and I.D. Adler (1994), "The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis", Mutat. Res., 341(2): 109-31.

Yamamoto, A., Hirouchi, T., Kawamorita, S., Nakashima, K., Sugiyama, A., & Kato, Y. 2017. Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells. Genes and Environment. 39: 22. doi: 10.1186/s41021-017-0082-z

Yauk, C.L., Y.E. Dubrova, G.R. Grant and A.J. Jeffreys (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus", Mutat. Res., 500(1-2): 147-56.

Yauk, C.L., M.J. Aardema, J. van Benthem, J.B. Bishop, K.L. Dearfield, D.M. DeMarini, Y.E. Dubrova, M. Honma, J.R. Lupski, F. Marchetti, M.L. Meistrich, F. Pacchierotti, J. Stewart, M.D. Waters and G.R. Douglas (2015), "Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays", Mutat. Res. Genet. Toxicol. Environ. Mutagen., 783: 36-54.

Yeat and J.C. Lin. 2015. Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes. Front. Genet., 6: 215. Doi: 10.3389/fgene.2015.00215.