For decades, toxicology has relied on a relatively small set of laboratory tests to understand how chemicals cause toxicity. Edgar Trelles Sticken, Advancing Toxicology Manager at Imperial Brands Science, explains what High Content Screening reveals about cellular responses.
These techniques can show the ‘what’, but not necessarily the ‘how’; for instance, the tests show what toxicity damage could arise, but not how or why it arose. This mechanistic understanding is vital for understanding the toxicological properties of chemicals.
High Content Screening (HCS) may hold the key to solving these problems and improving toxicology testing, especially with the continued rise and popularity of next generation products (e.g. vapes and nicotine pouches) as potential alternatives to cigarette smoking1.
Rather than reducing cellular behaviour to a single readout, HCS uses imaging-based analysis to examine how individual cells respond across multiple biological pathways within the same experiment. Fluorescent labelling and automated microscopy are combined with image analysis, allowing cellular stress and related changes to be observed at single-cell resolution.
That level of detail becomes important when cigarette smoke is used as a reference point. In-vitro exposure to cigarette smoke produces a strong oxidative stress signal, alongside activation of stress-associated pathways that respond to antioxidant intervention. Work published in Mutagenesis shows that oxidative stress plays a central role in cellular responses to cigarette smoke in advanced in-vitro systems.
Looking at cellular responses side-by-side
Laboratory studies using HCS have examined extracts generated from NGP alongside extracts derived from reference cigarette smoke. In human coronary artery endothelial cells, cigarette smoke extracts consistently trigger pronounced oxidative stress responses, which, amongst other factors, contribute to the development of atherosclerosis in the human body. These responses are accompanied by activation of stress-related signalling pathways, which are substantially reduced when cells are treated with N-acetylcysteine, a compound known to limit reactive oxygen species. This effect is documented in a scientific poster from Imperial Brands.
When non-combustible NGP are examined under the same conditions, a different pattern appears. For instance, extracts from tobacco-free nicotine pouches and from snus show minimal oxidative stress signalling. Where biological effects are detected, they occur only at much higher nicotine-normalised concentrations than those required to trigger responses from cigarette smoke extracts. Previous laboratory work shows lower biological activity for tobacco-free nicotine pouch extracts compared with both tobacco snus and cigarette smoke, as reported in a study published in Applied In Vitro Toxicology.
At this stage, interpretation matters more than detection. A measurable cellular response does not automatically imply comparable toxicological concern. The mechanism behind that response shapes how it should be understood.
Dose, exposure and why context matters
One reason laboratory findings can be difficult to interpret is dose. Cells grown in culture are often exposed to concentrations far higher than those encountered in everyday use, and responses at those levels can reflect physical stress rather than effects driven by specific chemicals.
In in-vitro settings like in HCS experiments, higher extract concentrations can be associated with increased osmolarity of the exposure medium. Under these conditions, cells show stress-related changes rather than effects linked to chemical toxicity.
Similar responses have been described in other areas of cell biology. For instance, this study published in Science Direct demonstrates that hyperosmotic conditions alone can alter signalling pathways and trigger programmed cell death – even in the absence of chemical toxicity.
This distinction matters when comparing different nicotine exposures. To make comparisons more meaningful, researchers assess biological activity on a nicotine-normalised basis, using the lowest concentration at which a response is observed. Viewed this way, cigarette smoke extracts show biological activity at far lower nicotine-equivalent levels than non-combustible NGP (i.e., cigarette smoke is far more potent than NGP aerosol).
Exposure outside the laboratory adds another layer of context. The nicotine concentrations at which cellular effects appear in HCS experiments exceed those typically reached by consumers. Clinical pharmacokinetic studies published in the Journal of Clinical Pharmacology show that nicotine levels reached during use of oral nicotine pouches remain well below concentrations associated with cellular effects observed in-vitro. Without that comparison, laboratory findings can appear more concerning than they are.
Making sense of complex results
HCS produces large amounts of data, which can be both a strength and a challenge. Making sense of those results depends less on collecting more information and more on how patterns are interpreted. In this example, researchers describe analytical frameworks that integrate dose-response information across multiple endpoints in Bioinformatics, offering structured ways to interpret complex biological datasets.
Consistency also matters. Studies using HCS rely on standardised cell models and controls, and inter-laboratory comparisons coordinated through groups such as CORESTA’s Next Generation Toxicity Testing Task Force have shown that similar platforms can produce comparable response patterns when those conditions are aligned.
HCS is not intended to replace existing regulatory toxicology tests, and it does not form part of routine regulatory requirements. Instead, it offers a different, mechanistical way of looking at the biological response. By focusing on how cells behave under different exposures, it adds further context where traditional tests may leave important questions unanswered.
As nicotine products continue to diversify, the challenge for toxicology is less about identifying effects and more about understanding what those effects mean. HCS does not resolve that challenge on its own, but it helps separate biological signal from experimental noise – a distinction that becomes increasingly important as exposure types move further apart.
For tobacco harm reduction science, progress depends on recognising where existing tools are sufficient to answer relevant questions, and where they are not. HCS falls into the latter category, not as a standalone solution, but as a means of revealing biological detail that would otherwise remain compressed into a single outcome.
This article draws on a Q&A with the author, alongside a scientific poster presenting the underlying data, both viewable on the Imperial Brands Science website.
