Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing

Abstract

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens

“…On the basis of the synthesis and critical review of the primary biomarkers identified and from relevant project work [ 67 , 68 , 69 , 74 , 214 , 215 ], the three CTA models are exemplified in the manner in which they can individually address the mechanisms and hallmarks of NGTxC as specified in the OECD IATA for NGTxC [ 3 ]. For the MIEs, the SHE CTA is able to address the cellular metabolism and receptor mechanisms of AhR signaling via CYP1B1, CYP2E1, epoxide hydrolase 1, GSH transferase and thioredoxin reductase.…”

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens

“…Figure 3 includes the most promising of the gene signalling markers that we identified. These can then be utilised for the assays or test methods being evaluated for the key events of the IATA in parallel work, as recently reported for epigenetic DNA methylation [ 30 ], gap junction [ 32 ], cell transformation [ 30 , 32 , 99 , 136 ], and similar work currently underway such as that for immune dysfunction (Corsini et al, paper in preparation), etc.…”

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens—a PARC project

“…In 2020, an OECD expert group developed an IATA for NGTxCs and published a consensus paper describing the overarching IATA, with the molecular initiating events (MIE) of cellular metabolism and receptor interactions, followed by the early key events (KEs) of inflammation and immune dysfunction, mitotic signaling, and cell injury, leading to (sustained) proliferation, morphological transformation and tumor formation ( Jacobs et al, 2020 ). Several assay evaluations and reviews have been conducted and published in relation to epigenetics ( Desaulniers et al, 2021 ), cell transformation ( Colacci et al, 2023 ), gap junction ( Sovadinova et al, 2021 ), gene signaling ( Oku et al, 2022 ), cell proliferation and oxidative stress ( Veltman et al, 2023 ). These publications provide details on the next steps needed to facilitate developments that will have regulatory relevance for the safety assessment of NGTxCs.…”

“…While several methods can be used to evaluate the dysregulation of GJIC, many have limited throughput and require specialized equipment ( Sovadinova et al, 2021 ). The SL-DT assay is the most commonly used in vitro method for evaluating the impact of toxicants or carcinogens on GJIC with a proven potential for high-content analysis and screening (HCA/HCS) ( Dydowiczova et al, 2020 ; Sovadinova et al, 2021 ). The SL-DT assay has been widely employed to study alterations in GJIC caused by hundreds of chemicals, including NGTxC.…”

“…The SL-DT assay has been widely employed to study alterations in GJIC caused by hundreds of chemicals, including NGTxC. Overall, the sensitivity of the SL-DT assay to predict IARC carcinogens (Group 1, 2A, or 2B) is 77% ( Sovadinova et al, 2021 ). However, most of the published data were generated using a rat liver epithelial cell line (WB-F344), and negative compounds (non-carcinogens) have not been widely assessed.…”