GenEvolutioN is pleased to present new findings on a miniaturised Cell Transformation Assay (CTA), enhanced by fluorescent labelling to improve accuracy and sensitivity in identifying non-genotoxic carcinogens. Developed using Bhas 42 cells—a 3T3 mouse fibroblast cell line transfected with the Ras gene—this method shows promise for food contact materials (FCMs), where non-intentionally added substances (NIAS) may pose hidden health risks.
1. Why Focus on Non-Genotoxic Carcinogens?
Not all carcinogens directly damage DNA (genotoxic); some act through epigenetic or indirect pathways, including:
- Altering cell signalling
- Increasing cell proliferation
- Affecting immune responses or apoptosis
In Food Contact Materials, non-intentionally added substances (NIAS) can migrate into food without ever being deliberately introduced in the manufacturing process. Detecting these hidden risks is critical for consumer safety and regulatory compliance.
2. How the Miniaturised CTA Works
Bhas 42 Cells
- Engineered fibroblast cells carrying multiple Ras gene copies.
- Pre-initiated to reveal early transformation steps.
Key Indicators of Transformation
- Basophilic staining and spindle-shaped cells
- Multilayer growth (cell piling) and random orientation
- Invasive outgrowth into the monolayer
Fluorescent Labelling with BMVC
- A novel staining approach that highlights transformed foci under fluorescence.
- Enables more accurate, consistent identification and quantitative analysis of cell transformation.
By utilising 96-well plates, researchers can assess cell transformation with less test material, saving both time and resources.
3. Combining Genomic Analysis with Fluorescent Labelling
GenEvolutioN’s enhanced CTA workflow includes:
- Transcriptomic Profiling : identifies up- or down-regulated genes related to carcinogenic processes (e.g., cell adhesion, apoptosis, immune response).
- BMVC Fluorescent Labelling : allows spectrophotometric detection of transformed cells, reducing reliance on manual observation.
- Automated Data Collection : ongoing efforts to integrate microplate washers/readers to streamline rinsing, foci detection, and data interpretation.
4. Applications for Food Contact Materials (FCMs)
Non-intentionally added substances (NIAS) in FCMs can pose long-term health risks if they exhibit carcinogenic properties. Standard genotoxicity tests may fail to detect indirect carcinogens. The enhanced CTA:
- Detects non-genotoxic carcinogens that conventional DNA damage assays might miss.
- Evaluates complex mixtures or extracts from packaging materials, identifying potential safety issues early.
- Aligns with EU regulations and global standards aiming to reduce unidentified chemical hazards in consumer products.
5. Looking Ahead: Automation & Broader Testing
The study proposes:
- Automation of Rinsing & Foci Analysis : leveraging microplate washers/readers to accelerate the assay workflow.
- Reduced Serum Variability : investigating non-fetal serum or lower serum mediums for consistent, reproducible results.
- Epigenetic Insights : exploring hypermethylation or hypomethylation changes in DNA compaction to catch additional non-genotoxic mechanisms.
This miniaturised, fluorescence-enhanced Cell Transformation Assay marks a significant step forward in detecting non-genotoxic carcinogens—especially crucial for Food Contact Materials, where hidden substances can migrate into consumer products. By combining advanced imaging and transcriptomic analysis, GenEvolutioN’s latest method offers high precision, reliability, and ethical testing alternatives to traditional animal studies.
References (Selected)
- Kirsch A. et al. (2020). Chem Biol Interact, 315:108900.
- Maeshima H. et al. (2009). Toxicol In Vitro, 23(1):148-57.
- Mascolo MG. et al. (2018). Carcinogenesis, 39(7):955-967.
- Ohmori K. et al. (2022). Int J Mol Sci, 23(6):3216.
- Rohrbeck A. et al. (2010). Toxicol Sci, 118(1):31-41.
