Genotoxicity

About

Genotoxicity describes the toxic effects of substances and material that cause genetic and epigenetic alterations. In many cases, those damages can be repaired by control and repair systems which detect alterations and correct it back to ensure normal cellular function. When damages cannot be repaired and when they last, genotoxic alterations become mutagenic and can even be passed to further generations when they occur in germ cells. Genotoxic alterations can be induced by a wide variety of compounds and materials. Examples include¹:

1. Benzene, whose metabolite 1-4-benzoquinone (BQ) constitutes a risk factor for hematopoietic cancers.
2. Pyrrolizidine alkaloids (PAs), which cause G:C -> T:A mutations.
3. Cigarette smoke components such as Nicotine, Nicotine-derived Nitrosamine KMetone (NNK) and Polycyclic Aromatic Hydrocarbons (PAHs) which induce cancerous effects by genotoxic gene point mutations, deletions, insertions, recombinations, rearrangements and chromosomal aberrations as well as epigenetic mechanisms such as alterations of cell proliferation and cell death functions including apoptosis inhibition.
4. Benzo[a]pyrene (BaP) which is another cigarette component and which is able to reduce the levels of global DNA methylation.
5. Nanomaterials (NMs) which can interact with lipids, proteins and nucleic acids and which might alter the charge of histone proteins which can lead to the alteration of chromatin organization. Nanomaterials have been defined by the European Commission as natural, incidental or manufactured materials which contain particles in an unbound, aggregated or agglomerated state and where 50% of the particles have the size of 1-100 nm in at least one spatial dimension.² Therefore, some NMs can pass various biological barriers and reach almost every location within an organism unhindered.
6. Many anticancer drugs such as alkylating agents, topoisomerase II inhibitors, and thiopurines can not only cause cell death but also induce sub-lethal mutations in normal somatic cells that can even give rise to secondary cancers. To evaluate genotoxicity during cancer therapy, the Phosphatidylinositol Glycan class A gene (PIG-A) assay is performed where PIG-A loss of functions are investigated.

Especially with the recent advances in nanotechnology where new products are released very frequently and where developments include applications also in medical solutions, it is a great aim to be able to assess the risk of chemicals and materials in concern of their potential genotoxic effects at an early stage during product development. There are tests which are widely used as standard tests to assess this risk¹. In the following, we list some available tests.

Short Term Tests (STTs)

Ames test (bacterial reverse mutation assay)

During this test, a histidine-auxotrophic Salmonella typhimurium mutant strain is seeded on petri plates and exposed to the test chemical. The number of colonies with histidine-auxotrophic -> histidine-prototrophic mutations is taken as a marker for the mutagenicity capacity of the test compound. This test is cost effective and simple but does not reflect the effect of test compounds in mammals which have a much more complex repair system than Salmonella typhimurium.

in vivo cytogenetics tests, which determine chromosome abnormalities such as aneuploidy and structural abnormalities.³

in vivo micronucleus assays (MNA) to screen for chromosomal damage

Micronucleoli (Howell-Jolly bodies) were first described in erythrocytes as a marker for cytogenetic damages. It was also shown that some viruses can increase the MN frequency, which can be taken as an indicator for the genotoxic potential of a test compound after exposure to the cell. The assay is fast, reliable and easy and is widely used in epidemiology to study cytogenetic damages and in the virology field.

in vivo transgenic animal models such as transgenic mouse model assays which are used for mutagenicity evaluation.

Next-generation sequencing technology, which detects mutagenic alterations.

Other in vivo test

comet assay (CA), for detecting DNA damages

During the comet assay, single cells are lysed in an agarose gel and a single cell gel electrophoresis is applied. The stained DNA will give a comet-like pattern which differs between different DNA alterations. The fluorescent intensity and the length of the comet are taken to estimate the extent of DNA damage. There is the neutral method (pH 8.4) for double stranded breaks where the variability is very high dependent on experimental factors such as agarose concentration, voltage gradient etc. and the alkaline method (pH > 13) for double and single strands. The comet assay is also proposed to assess the levels of genetic damages in human diseases, like essential hypertension, chronic kidney disease (CKD), type 2 diabetes, and even in cancer chemoprevention.

Figure 1 summarizes the current available assays which are used for genotoxicity risk assessment.

Figure 1: Current approaches for genotoxicity risk assessment (Ren et al. 2017)¹.

Genetic defects can have a significant impact on biological mechanisms leading to severe diseases or even to death. Unfortunately, many of those mechanisms have remained unclear until today and it is important to keep investigating the molecular functions that lead to genotoxicity. The new knowledge will allow us to define new genotoxicity endpoints and to develop new approach methods to fit the current needs of integer risk assessment in concern of genotoxicity and to improve the current approaches which we have with the above mentioned available assessment assays. Some approaches include¹:

Germ cells

Germ cells are important to investigate since they are the cells which carry mutations along to further generations but testing has been challenging since germ cell impairment was predicted based on the effect of a substance on somatic cells. New assays are needed and novel assays were developed to specifically investigate chromosomal damages in germ cells, including the sperm and pedigree tandem repeat mutation analysis⁴, high-throughput screening⁵ and DNA microarrays⁶.

With those new methods it is possible to measure new endpoints such as chromosomal aneuploidies and structural aberrations, copy number variation (CNV) and single nucleotide variant (SNV), tandem repeat mutations, insertions/deletions and mutations in non-coding sequences such as repetitive elements.

Stem cells

There are different types of stem cells. They can be induced by expressing transcription factors in somatic cells to give rise to induced pluripotent stem cells (iPSC) or they can be taken from embryos, called Embryonic Stem Cells (ESC) for example. Stem cells have many benefits. One of them is the fact that they are more protected against genetic instabilities when compared to somatic cells due to their more effective DNA repair mechanisms. Due to their beneficial properties they are aimed to be used for medical treatments such as more effective transplantation medicine where transplants might get less rejected. Nevertheless, it has been shown that some stem cells are able to avoid apoptosis, leading to cancer cells, and that they are prone to have more point mutations than expected. Therefore, it is a great aim to develop new methods which allow the generation of safe stem cells which have a limited amount of mutations. Approaches might include a full classification of known functional mutations and suitable biomarkers, or the suicide gene strategy where stem cells are marked with a suicide gene that can be activated to kill all stem cells when required during therapy.

Genetic toxicology and epigenetics

Genetic toxicology and epigenetics investigates altered DNA methylation (hyper and hypo-methylation as a hallmark of cancer), histone modification (methylation, acetylation, phosphorylation, sumoylation and ubiquitination) as well as non-coding RNA and chromatin remodeling.

Based on this knowledge, new epigenetic drugs are being designed such as DNA methylation inhibitors. Since they are directly affecting genetic heritage, methods need to be developed with which it is able to assess the risk of such drugs.

Further assessment approaches

Further assessment approaches take into account endpoints such as DNA methylation, histone modification degrees and enzymatic activity of DNA methyltransferases to evaluate drug’s actions and efficacy and also include new imaging approaches. New imaging technologies for high resolution optical imaging includes:

1. Confocal laser scanning
2. Two photon excitation microscopy
3. High content cell imaging
4. Digital tissue scanning
5. 3D quantitative DNA methylation imaging (3D-qDMI)
6. Detection of micronuclei (MN)

Thereby, the detection of micronuclei is a great tool to investigate genetic alterations as it is summarised in figure 2. Nevertheless, the exact relation between genetic alterations and the formation of micronuclei still has to be investigated and new methods have to be developed.

Figure 2: An overview over mechanisms that lead to micronuclei formation. (Ren et al. 2017)1.

To face the challenges which come along with genotoxicity risk assessment, the International Workshop on Genotoxicity Testing suggests the use and integration of available data in mathematical and computational models to be able to characterise the dose-response relationship in a quantitative risk assessment approach.¹

At SaferoWorldbyDesign we offer this integrated approach based on new technologies to assess the genotoxic risk of chemicals and materials. Thereby, we integrate new in vitro technologies with computational modelling, which take into consideration already existing data in a tiered strategy. This service is underlined by a profound knowledge and data infrastructure based on highest standards which allows us to organise your data in a safe and FAIR manner. Do you want us to develop a solution which fits your specific needs and interests? Please feel free to reach out and to contact us!

Your SaferWorldbyDesign Team

References

(1) Ren, N.; Atyah, M.; Chen, W.-Y.; Zhou, C.-H. The Various Aspects of Genetic and Epigenetic Toxicology: Testing Methods and Clinical Applications. J Transl Med 2017, 15 (1), 110. https://doi.org/10.1186/s12967-017-1218-4.

(2) European Commission. (2011). Commission Recommendation of 18 October 2011 on the definition of nanomaterial. Retrieved on 12.07.2022 from https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32011H0696

(3) Ozkan, E.; Lacerda, M. P. Genetics, Cytogenetic Testing And Conventional Karyotype. In StatPearls; StatPearls Publishing: Treasure Island (FL), 2023.

(4) Ralf, A.; Montiel González, D.; Zandstra, D.; Van Wersch, B.; Kousouri, N.; De Knijff, P.; Adnan, A.; Claerhout, S.; Ghanbari, M.; Larmuseau, M. H. D.; Kayser, M. Large-Scale Pedigree Analysis Highlights Rapidly Mutating Y-Chromosomal Short Tandem Repeats for Differentiating Patrilineal Relatives and Predicting Their Degrees of Consanguinity. Hum Genet 2023, 142 (1), 145–160. https://doi.org/10.1007/s00439-022-02493-2.

(5) Macarron, R.; Banks, M. N.; Bojanic, D.; Burns, D. J.; Cirovic, D. A.; Garyantes, T.; Green, D. V. S.; Hertzberg, R. P.; Janzen, W. P.; Paslay, J. W.; Schopfer, U.; Sittampalam, G. S. Impact of High-Throughput Screening in Biomedical Research. Nat Rev Drug Discov 2011, 10 (3), 188–195. https://doi.org/10.1038/nrd3368.

(6) DNA Microarray Technology Fact Sheet. Genome.gov. https://www.genome.gov/about-genomics/fact-sheets/DNA-Microarray-Technology (accessed 2023-08-16).

Our services

Our solutions at SaferWorldbyDesign are based on a tiered strategy approach including new in vitro technologies combined with machine learning QSAR modelling developments. Thereby:

1. We determined precise biotransformation capacities of different human cell lines which are used for genotoxicity studies such as HepG2, HepaRG, ACHN and LS74T which can be used for genotoxicity characterization.
2. We have also developed a new genotoxicity assay based on histones H2AX and H3 quantification which allows to discriminate efficiently aneugens, clastogens and cytotoxic compounds in all cellular models.
3. We also offer the Ames microplate format (MFP) assay, which is a miniaturized, liquid version of the plate agar Ames tests performed in 384-well plates that takes advantage of a color change-based readout.

For further information please see the content on genotoxicity testing:

Together with our partner Preditox we further offer the PrediScreen assay, which is performed on human cells and which makes it possible to study the carcinogenic potential of any type of compound or of samples according to 3R principles. The test allows to investigate whether:

1. The test compound is cytotoxic or not.
2. The test compound is genotoxic or not.
3. The genotoxic mode of action involved is a clastogenic mode of action.
4. The genotoxic mode of action involved is an aneugenic mode of action.
5. The compound needs bioactivation to be genotoxic.

For more information please see the content on our Peritox solution:

If you want us to develop a customized solution for you also regarding knowledge infrastructure, data management and modelling, please feel free to reach out to us.