Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept

Yvette Dirven, Dag Markus Eide, Erika Witasp Henriksson, Rune Hjorth, Anoop Kumar Sharma, Anne Graupner, Gunnar Brunborg, Jarle Ballangby, Anne Mette Zenner Boisen, Stellan Swedmark, Kristine Bjerve Gützkow, Ann Karin Olsen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ-cell specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through 1) visual inspection of DNA content distributions, 2) setting DNA content thresholds, and 3) modelling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.
Original languageEnglish
JournalEnvironmental and Molecular Mutagenesis
Issue number2
Pages (from-to)88-104
Number of pages17
Publication statusPublished - 2023

Bibliographical note

Funding was received from the Nordic Working Group for Chemicals, Environment, and Health, under the Nordic Council of Ministers (2021-022,2021-101). This publication was funded by the Nordic Council of Ministers; however, the content does not necessarily reflect the Nordic Council of Ministers' views, opinions, attitudes, or recommendations. Financial support was also received from Swedish Chemicals Agency and the Research Council of Norway through its Centers of Excellence funding scheme, project number 223268/F50 CERAD.


  • Genotoxicity
  • OECD TG 489
  • Primary spermatocyte
  • Round spermatid
  • Total fluorescence intensity


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