LITMIR.BIZ

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or cell type (Clark et al. 2014; Gu and Kay 2010; Lu and Clark 2012). Thus, alterations in miRNA amounts, even if minute, could feasibly have a large impact on transcriptional profiles. The consequence is important in terms of biological effect, as well as for the identification of biomarkers of health impact.

      The need for specific and sensitive biomarkers spans research and development, regulatory, safety, and clinical sectors. Biomarkers can indicate exposure, biological effect, and sensitivity. All three areas have important purposes and applications. Those that measure biological effect more directly link to biological function, and thus to the putative adverse health effects that a perturbation may mediate. A biomarker may be a measurable alteration—chemical, biochemical, physiological, behavioral, or of some other kind—within an organism (World Health Organization and International Programme on Chemical Safety 1993). miRNAs have the potential to be good biomarkers of biological effect because they are well defined, chemically uniform, restricted to a manageable number, and stable (not readily degraded). They also get released into extracellular matrices, where they are accessible and measurable. In these biofluids such as blood, urine, and sputum, miRNAs serve as unique biomarkers for a minimally invasive prediction of toxicant exposure. The altered biological pathways that is consequent upon (and due to) miRNA changes can therefore reflect the mechanisms of toxicant-related diseases. Measurements of these miRNAs in biofluids can therefore serve as biomarkers of effect.

Indeed, changes in intracellular miRNA expression due to toxicant exposure have been well documented in multiple tissues and model organisms (Yu and Cho 2015) and are regulated by both genetic and epigenetic mechanisms. DNA hypo- and hypermethylation and histone modifications are involved in the regulation of the expression of miRNA promoters (Tomasetti et al. 2019). As a high proportion of miRNAs are embedded in CpG islands susceptible to methylation, miRNA genes are methylated more frequently than protein-coding genes (Kozomara et al. 2019; Morales et al. 2017). There is also evidence that some miRNAs can interact with other miRNAs, in a form of self-regulation (Hill and Tran 2021). In addition, mechanisms for the alteration of intracellular miRNA expression through exposure to environmental carcinogens have been proposed that involve the modulation or blocking of normal miRNA processing and maturation (Izzotti and Pulliero 2014). DNA damage can alter miRNA expression via p53-dependent mechanisms. p53 interaction with the Drosha/DGCR8 processing complex modulates the processing of pri-miRNAs to pre-miRNAs. In addition, Dicer, another processing unit that produces the final mature miRNA, is a direct transcriptional target for p53. Alternatively, the binding of electrophilic metabolites to nucleophilic sites of miRNA precursors can form miRNA adducts that are not able to access the catalytic pockets of Dicer, therefore arresting miRNA maturation. Further, toxicant metabolites binding to Dicer itself in the proximity of miRNA catalytic sites can block the maturation of miRNA precursors (Izzotti and Pulliero 2014).

      The interest in miRNAs as biomarkers of exposure to environmental toxicants and resultant biological effects is bolstered by their stable detection in extracellular biofluids, where they can be non-invasively sampled. There are a number of mechanisms by which miRNAs are released from the cell into these matrices (Condrat et al. 2020) and, importantly, can be linked to tissue or cell type specificity (or both), as well as to mechanisms of biological perturbation that may relate to stress response, toxicity, and disease. In this chapter we review the promise of these putative biomarkers, together with the technical challenges that lie ahead if we want to establish them in the practice of toxicology and regulatory sciences.

      Mechanisms that Contribute to Extracellular miRNA Release

      When using the miRNAs present in biofluids as biomarkers of tissue perturbation, toxicity, or disease, one must understand the origin of these non-coding RNAs in the extracellular space. Most studies have focused on the miRNAs found in blood and urine, although extracellular miRNA has been noted in sputum, tears, amniotic fluid, cerebrospinal fluid, breast milk, and bronchial lavage fluid, among other sites (Arroyo et al. 2011; Chen et al. 2008; Turchinovich et al. 2011; Valadi et al. 2007; Vickers et al. 2011; Wang et al. 2010a; Weber et al. 2010). Although many miRNAs seem to be ubiquitously present in many characterized biofluids, blood plasma has the highest amount of uniquely present miRNAs (Weber et al. 2010). Blood is a complex liquid “tissue” that interacts with many cell types not accessible to other fluids, including those of hematopoietic residence—predominantly erythrocytes and reticulocytes. The contributions from these cell types are important to characterize, so that they can be separated, if needed, from the signals of other cell types or tissues. For example, in whole blood samples, miRs-486-5p and -451a are highly represented because they are derived from erythrocytes and can complicate or mask the evaluation of other putative biomarker miRNAs of lower abundance in the blood (Juzenas et al. 2020). Mechanistically, these potentially interfering factors can be removed by processing blood into serum or plasma, or, in cases where hemolysis may increase the proportion of erythrocyte-derived miRNAs in serum, blocking steps can reduce the measurement of these miRNAs (LaBelle et al. 2021).

Extracellular miRNAs are released by their cells of origin through a number of different mechanisms, which can simply be classified into “active” and “passive” release (Harrill et al. 2016). Those that are passively released are due to mechanisms of plasma membrane breakdown that lead to the spilling or “leaking” of intracellular contents into proximal biofluids. This can occur with cell death, necrosis, or toxicity. Importantly, some miRNAs have cell- or tissue-specific expression (Bailey and Glabb 2018). As such, these biomarkers may indicate specific tissue damage or disease. A number of studies have demonstrated the specificity of miRNAs and other non-coding RNAs for different tissues in clinical and non-clinical species such as human (miRNA TissueAtlas: Ludwig et al. 2016), rat (RATEmiRs database: Bushel et al. 2018; Smith et al. 2016), mouse (Isakova et al. 2020), and dog (Koenig et al. 2016). According to various analyses of these resources, those considered tissue-specific ranged from ~ 100 to ~ 400 miRNAs, depending on the species, and there was noted overlap among mammals that indicated putative biomarkers that could be used for cross-species profiling (Figure 2.1).

      Packaging into Vesicles and Mechanisms of Release

      Figure 2.1 Tissue-specific miRNAs in mammals. Human tissue-specific miRNAs that were described as part of the human miRNA tissue atlas study (Ludwig et al. 2016) (light grey circle with dotted/dashed line) were cross-referenced with miRNA atlas studies that examine tissue-specific miRNAs in mouse (medium grey circle with dashed line) and rat (dark grey circle with solid line). Those miRNAs that transverse all three circles are ideal cross-species biomarkers for toxicological studies.

Both prokaryotic and eukaryotic cells release membrane-enclosed microvesicles that package a variety of different cellular-derived components, including nucleic acids, proteins, and lipids. The vesicles range in size (from small 50–150 nm exosomes to large 1,000–10,000 nm tumor cell-derived oncosomes), location of biogenesis, and content. These vesicles play important roles in cellular communications and their content is enriched in small non-coding RNAs (such as miRNAs, lncRNAs, snoRNAs, piRNA, snRNAs, etc.), but also in intact mRNAs and in fragmented tRNAs, mRNAs, lncRNAs, rRNAs, and other nucleic acids (Turchinovich et al. 2019). The roles of these packaged contents are not entirely clear for all RNA species; however, many studies have demonstrated that some of these RNAs can mediate differential responses in recipient cells. Many studies have focused on miRNAs in this paracrine role (reviewed in O’Brien et al. 2020) which therefore may make them serve as putative biomarkers of “active” release due to homeostatic, responsive, and perturbed cellular states. A number of characterization studies have been performed to determine the RNA contents of extracellular microvesicles isolated from human plasma, saliva, and urine, as well as cell lines of different lineages (see reviews in Amorim et al. 2017 and Turchinovich et al. 2019). In one of the first studies that use deep sequencing to profile extracellular vesicle (EV) RNAs, small RNAs were reported to dominate these “shuttle”-derived fractions; however, the distribution was different from that of the parent mouse dendritic cells co-cultured with cognate T cells (Nolte-’t Hoen et al. 2012). Importantly,

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