Workshop Report 32

Cellular adaptation: Relevance for risk assessment, physiological interpretation: Phenotypic anchoring

Emma Marczylo, PHE, UK

The vast majority of the mammalian genome is composed of so-called noncoding DNA, with only approximately 1‑2% of the mammalian genome coding for proteins. The majority of noncoding DNA is transcribed into ncRNAs, which play major roles in regulating gene expression. Whilst lncRNAs do this in a variety of ways, including chromosome remodelling and transcriptional or post-transcriptional regulation, short ncRNAs predominantly regulate gene expression at the post-transcriptional level. NcRNAs interact with epigenetic mechanisms to form a robust regulatory epigenetic network (Marczylo et al, 2016). Many ncRNAs, particularly miRNAs, have been shown to be involved in both toxicity and disease. Specifically, ncRNA play a central role during cellular adaptation in response to changes in the environment. Following an environmental exposure, cellular processes are activated in an attempt to regain homeostasis. These processes may be transient, returning to the original state once the insult is removed, or they may become established as the ‘new homeostasis’. Such adaptations may be positive, providing protection against further stress (Wheeler and Jong, 2007; Jain et al, 2014); or they may be negative, resulting in an increased susceptibility to further stress (Greathouse et al, 2012; Wong et al, 2015). If cells are unable to adapt to an environmental exposure, adverse effects may evolve that may either be reversible (leading back to homeostasis) or irreversible which ultimately results in cell death. For RA, it is vital to distinguish between cellular adaptation and adverse effects.

In determining cellular ‘normality’ with the aim of making use of ncRNAs for RA, it is important to understand and characterise the variability in ncRNAs both within individuals (tissue specificity) and between different individuals and different species that are present in the same steady state (normal environment). For instance, more than 500 different miRNAs were present in the plasma of 18 disease-free human volunteers of which approximately 50 miRNAs were present in all samples. Further, 10 of the most highly expressed miRNAs accounted for 90% of the total miRNA expression, and 5 of these were blood cell-associated. Finally, 10 of the most stably expressed miRNAs included 5 of the most highly expressed ones (Tonge and Gant, 2016). Variability in ncRNA expression should also be understood in response to changes in the normal environment that do not exert adverse effects both within individuals and between individuals. This will enable identification of genetic susceptibilities, normal ranges of ncRNA expression, and adaptations with increased susceptibilities.

In determining the relevance of miRNA-initiated cellular adaptations to environmental stressors, it is important to distinguish whether the change in ncRNA expression is the cause or the consequence of an apical effect. Yet, whilst a robust, dose-dependent relationship between specific ncRNA(s) and environmental stressor(s) or subsequent effect(s) is vital for RA, establishing causality is not necessarily essential. ncRNAs that do not in themselves directly induce adverse effect(s), but instead act as markers of exposure and/or predictors of future toxicity, may also be useful in regulation (Marczylo et al, 2016). Increasingly, the potential use of miRNAs as biomarkers is being investigated since they are secreted in multiple body fluids (including blood, semen, saliva), and they are stable (secreted within exosomes), accessible, and easily measured (Gant et al, 2015). Spermatozoal miRNAs are of particular interest for regulatory purposes since they can be altered by environmental exposures, transmitted across generations, involved in the physiological or pathophysiological development of subsequent progeny and are easily collected and analysed (Liu et al, 2012; Marczylo et al, 2012; Rodgers et al, 2013, 2015; Gapp et al, 2014; Stowe et al, 2014). To be used as biomarkers, ncRNAs should further be sensitive, specific, and linearly related to exposure and effect.

With regard to the physiological interpretation (i.e. phenotypic anchoring) of alterations in ncRNA expression, these molecules pose unique challenges since they act at multiple levels forming part of an interactive network. For example, miRNAs have multiple mRNA targets, just as potential mRNA targets are targeted by multiple miRNAs. Consequently, the interpretation of observations may be challenging. To facilitate phenotypic anchoring, ncRNA expression profiles may be correlated with other profiles and phenotypic endpoints (Akinjo et al, 2016). In this respect, ‘-omics’ technologies allow simultaneous profiling of multiple variables using a systems biology approach.

In conclusion, ncRNAs are important regulators of gene expression and represent novel mechanisms and markers of toxicity that might be useful for regulatory purposes. To explore such use, a greater mechanistic understanding should be obtained, e.g. by performing additional analyses on surplus biological samples from existing regulatory studies, thereby avoiding the use of extra animals. It may also be considered to adapt existing testing guidelines to incorporate ncRNA analyses, as appropriate, to begin collecting data on ncRNA in a regulatory context.


Considering the possible use of miRNAs as indicators for exposure in live animals: How well established is the correlation between alterations recorded in the blood, urine or other body fluids and changes seen in target organs? – Many studies have investigated plasma miRNAs as markers of tissue/organ-specific toxicity. Nevertheless, to date, there is very little information regarding the variation of expression in a given biological fluid and how this expression alters in response to factors such as age, gender, obesity and smoking status. Indeed, a number of studies have reported correlations between organ-specific toxicities and miRNAs that are actually predominantly associated with blood cells. Therefore it may be questioned whether these correlations are instead a consequence of blood sample preparation. Thus, robust correlations have yet to be established.

Is it possible to observe changes in gene expression before histopathological alterations can be observed, and are there changes in gene expression that are unrelated to histopathological findings? – There is a large collection of literature on gene expression changes at sub-toxic doses, which will include changes that do not directly relate to the ultimate apical effects. To determine the relevance of such expression changes, it is important to understand mechanisms of toxicity and to perform full dose-responses.