Ann-Karin Olsen

Heritable mutations in male germ cells pose a critical risk to human health and future generations, however standardized methods for assessing germ cell genotoxicity remain limited. We refined the in vivo alkaline comet assay (proof-of-concept (Dirven et al. 2023); protocol (Olsen et al. 2024)) to detect DNA damage in testicular germ cells, with selective addressment of haploid spermatids and primary spermatocytes. Measurements of DNA damage (% Tail DNA) and DNA content (total fluorescence intensity) in individual comets were combined with visual comet identification to distinguish testicular comet populations based on differences in DNA content and appearance. To verify the method’s functionality and reliability, DNA damage was assessed in rats exposed to the direct-acting, well-characterized genotoxicants X-rays and ethyl methanesulfonate across distinct testicular cell populations, alongside liver and blood. To minimize experimental variation, the protocol included stringent standardization of animal handling, tissue processing, and comet assay procedures. Both X-rays and EMS induced significant DNA damage in testicular germ cells, with comparable responses across testicular cell types and similar (X-rays) or higher levels observed in somatic tissues. The low inter-animal variability observed supports the robustness of the method. Importantly, inclusion of testicular germ cells in OECD test guideline 489 would provide a valuable tool for hazard identification and mutagenicity classification of chemicals under the Globally Harmonized System of Classification and Labelling of Chemicals. This versatile, sensitive, and resource-efficient assay enhances the assessment of male-mediated genetic risks and supports regulatory efforts to protect reproductive health and safeguard the genetic integrity of future generations through the use of safer chemicals.

Current genotoxicity testing strategies face several challenges, including a high incidence of misleading positive results that lead to unnecessary animal testing, limited mechanistic insights, insufficient integration of innovative methodologies, and a lack of quantitative assessment. Despite rapid advancements in technology and scientific understanding, genotoxicity testing batteries have remained largely unchanged for years. To modernize genotoxicity assessment and incorporate innovative approaches, the development of Integrated Approaches for Testing and Assessment (IATAs) is essential. These frameworks combine existing knowledge with data from New Approach Methodologies (NAMs) aiming to reduce or eliminate reliance on in vivo testing. Genotoxicity is particularly well‐suited for IATA development as numerous cutting‐edge, non‐animal methods have emerged in recent years, including 3D test systems, Prediscreen, MultiFlow, ToxTracker, and transcriptomic‐based biomarkers such as GENOMARK and TGx‐DDI. However, the integration of NAMs into IATAs must be systematic and scientifically robust. In this process, the Adverse Outcome Pathway (AOP) framework plays a crucial role by linking molecular‐level events to adverse health effects, thereby supporting the structured selection of NAMs. This article explores the key challenges and gaps within the current European regulatory frameworks for chemical compound genotoxicity assessment and discusses how an AOP‐based IATA can address these issues. Additionally, we present a global AOP network for permanent DNA damage, designed to guide IATA development and improve regulatory decision‐making. This integrated approach has the potential to enhance the accuracy, efficiency, and ethical standards of genotoxicity assessment while reducing reliance on animal testing.

Enniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.

Few protocols are available for retrieving male germ cell-specific information on DNA damage dynamics or assessing the potential harmful effects of environmental contaminants, drugs, or lifestyle factors related to genotoxicity. Here, we present a protocol for evaluating testicular germ cell genotoxicity using a modified version of the alkaline comet assay in rodent testicular germ cells. The protocol includes experimental design, preparation of testicular cell suspensions, comet analysis, germ cell-specific scoring, and data curation methods to collect information on testicular male germ cells, specifically spermatids and spermatocytes. For complete details on the use of this protocol please refer to Olsen et al., in preparation. Highlights: -DNA damage levels can be specifically measured in testicular germ cells using the developed revised version of the alkaline comet assay -1C spermatids as well as 4C primary spermatocytes can be assessed -Both manual- and modeling-based approaches were developed that facilitate user-friendly protocols to select 1C spermatid comets. -This protocol expands the limited methodologies available to study germ cell DNA damage dynamics

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1^−/−Neil2^−/− mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1^−/−Neil2^−/− mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.

MicroRNAs (miRNAs) have been linked to several diseases and to regulation of almost every biological process. This together with their stability while freely circulating in blood suggests that they could serve as minimal-invasive biomarkers for a wide range of diseases. Successful miRNA-based biomarker discovery in plasma is dependent on controlling sources of preanalytical variation, such as cellular contamination and hemolysis, as they can be major causes of altered miRNA expression levels. Analysis of plasma quality is therefore a crucial step for the best output when searching for novel miRNA biomarkers. Plasma quality was assessed by three different methods in samples from mother-child duos (maternal and cord blood, N = 2x38), with collection and storage methods comparable to large cohort study biobanks. Total RNA was isolated and the expression profiles of 201 miRNAs was obtained by qPCR to identify differentially expressed miRNAs in cord and maternal plasma samples. All three methods for quality assurance indicate that the plasma samples used in this study are of high quality with very low levels of contamination, suitable for analysis of circulating miRNAs. We identified 19 significantly differentially expressed miRNAs between cord and maternal plasma samples (paired t-tests, FDR±1.5), and we observed low correlation of miRNA transcript levels between cord and maternal samples throughout our dataset. Our findings suggest that good quality plasma samples suitable for miRNA profiling can be achieved from samples collected and stored by large biobanks. Incorporation of extensive quality control measures, such as those established here, would be beneficial for future projects. The overall low correlation of miRNA expression between cord and maternal samples is an interesting observation, and promising for our future studies on identification of miRNA-based biomarkers in cord blood plasma, considering that these samples were collected at term and some exchange of blood components between cord and maternal blood frequently occur.

Mutagenesis, Volume 33, Issue 1, 1 January 2018, Pages 25–30, https://doi.org/10.1093/mutage/gex039