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release of miRNA into the circulation. In the canonical pathway, miRNAs encoded within genes are transcribed by RNA Polymerase II (Pol II Transcription) into large primary miRNAs. Primary miRNAs are cleaved within the nucleus by the Drosha Microprocessing Complex (Drosha Complex Processing), which gives rise to precursor miRNAs. Precursor miRNAs are exported to the cytoplasm by Exportin 5. They are next cleaved by Dicer, followed by loading into RISC. The RISC facilitates removal of the passenger strand RNA to generate mature miRNA. Mature miRNAs can be actively secreted into circulation in association with RNA-binding carrier proteins (AGO2/HDL/LDL), or by packaging into microvesicles/exosomes (not shown). Alternatively, mature miRNA targets cognate mRNA in the cytoplasm through complimentary base pairing, which leads to regulation of gene expression.

      Mature miRNAs regulate their target mRNAs by binding to 3ʹ untranslated regions (3ʹUTR) of mRNAs; however, miRNA binding sequences have also been identified within 5ʹ UTRs (Friedman et al. 2009). Mature miRNAs are guided to their cytosolic target mRNAs by complementary base pairing, and miRNA targeting is reliant on base pairing of the miRNA seed region, nucleotides 2-7, to mRNA targets. Perfect miRNA-mRNA base pairing leads to the degradation of the target mRNA, whereas imperfect base pairing leads to decreased mRNA translation, which can be restored once the repressing miRNA is degraded. Intriguingly, it has also been shown that miRNA-mRNA interaction with the 5ʹ UTR can increase the translation of the mRNA, and that some miRNAs can target promoters or enhancers (Fabbri 2018; Odame et al. 2021; Vasudevan et al. 2007). Biological functions of separate miRNAs have been extensively studied using a variety of miRNA-knockout/knockdown models and transgenic overexpression experiments (Hammond 2015; Saliminejad et al. 2019).

      Circulating miRNAs as Biomarkers for Disease

      miRNA expression profiling has demonstrated dysregulation of miRNA expression in a wide range of human diseases (Bonneau et al. 2019). Dysregulated miRNA expression has been shown to affect a large variety of cellular processes, for example transcription, signal transduction, cellular proliferation, differentiation, apoptosis, and epithelial-mesenchymal transition (Aleckovic and Kang 2015). Alterations in miRNA expression signatures offer great potential for use as clinical biomarkers, because miRNAs are stable in biological samples, including formalin fixed paraffin sections (FFPE) and plasma (Amini et al. 2017; Aryani and Denecke 2015; Heneghan et al. 2010; Kotorashvili et al. 2012; Wimmer et al. 2018). Additionally, miRNAs can be reliably detected in multiple bodily fluids such as blood, serum, amniotic fluid, breast milk, bronchial lavage, cerebrospinal fluid, colostrum, peritoneal fluid, plasma, pleural fluid, saliva, seminal fluid, tears, and urine (De Guire et al. 2013; Hammond 2015; Lawrie et al. 2008; Weber et al. 2010). But the total RNA concentration and the total number of distinct miRNAs can differ vastly between various body fluids (Weber et al. 2010). Likewise, specific circulating miRNAs can be enriched within specific fluids.

      Circulating miRNAs are released into circulation in one of two forms: they are either bound to specific RNA-binding proteins (Ago; high density lipoprotein, HDL; or low-density lipoprotein, LDL) or encapsulated in microvesicles along with proteins, lipids, and other nucleic acids (Aleckovic and Kang 2015; Cui et al. 2019; Mori et al. 2019; see Figure 4.2). The release mechanisms of protein- or lipid-bound miRNAs is largely unknown, whereas exosomal miRNAs are known to be selectively recruited and actively secreted in a regulated manner (Aleckovic and Kang 2015). When injected intravenously, the exosomes can remain in the circulation for up to two hours, which implies a large range in the bioavailability of circulating miRNAs (Mori et al. 2019).

The potential use of circulating miRNAs as non-invasive diagnostic and prognostic biomarkers for disease status in biological fluids was first realized in the field of cancer biology because cancer diagnosis and prognosis rely on invasive tissue biopsies (Correia et al. 2017; Iorio and Croce 2012). More recently, changes in circulating miRNA profiles upon heavy metal exposure have been described. In consequence, this chapter strives to provide a comprehensive resource, which will highlight studies that have identified circulating miRNAs as a result of heavy metal exposure and assist with rigor and reproducibility in future studies. We have chosen to limit our focus to the five metals (arsenic, lead, mercury, cadmium, chromium) ranked currently at the top of the ASTDR substance priority list on the basis of their significant toxicity and of the high potential for human exposure to them.

      Circulating miRNAs Associated with Arsenic Exposure

      Arsenic (As) is a naturally occurring metalloid ubiquitously distributed throughout the earth’s crust and groundwater. It is also found in the atmosphere and in food, particularly in cereal and cereal products (Cubadda et al. 2017; Zhang et al. 2020). Arsenic is classified as a Group I human carcinogen by the International Agency for Research on Cancer, because chronic exposure to arsenic is strongly associated with an increased risk for cancer development in skin, urinary bladder, and lung in humans (IARC 1980, 2012; Straif et al. 2009). Therefore arsenic contamination is considered a serious global public health issue.

      Arsenic contamination affects several countries, for example Bangladesh, India, Taiwan, China, Ghana, United States, Argentina, Vietnam, and Chile (Hunt et al. 2014). An estimated 225 million people are chronically exposed to arsenic from drinking water, at concentrations that exceed the maximum contaminant level (MCL) of 10 μg/L recommended by the World Health Organization (WHO) (Podgorski and Berg 2020). In Bangladesh alone, arsenic levels exceeding 50 µg/L have been reported, affecting between 21% and 48% of the total population (Rahman et al. 2001). In the United States, approximately 3 million people relying on private domestic wells for water usage are exposed to high concentrations of arsenic (Ayotte et al. 2017). In addition to naturally occurring arsenic, human-made arsenic-based compounds constitute other sources of arsenic exposure. Arsenic-based pesticides are still widely used in agriculture (Li et al. 2016); in this way they produce significant arsenic contamination in the environment and contribute to a number of acute and sub-acute arsenic intoxication cases (Armstrong et al. 1984; Li et al. 2016).

      The main forms of arsenic present in the environment are arsenate (iAs^V), arsenite (iAs^III), and other organic arsenicals such as methylated arsenicals—monomethyl arsenic acid (MMA^V) and dimethyl arsenic acid (DMA^V) (Thomas 2015; Tsuji et al. 2019). After ingestion, arsenate or arsenite are readily absorbed in the gastrointestinal tract and transported through the blood to other parts of the body. In the liver, around 90% of arsenic undergoes a series of sequential reduction and oxidative-methylation reactions. iAs^V is reduced to iAs^III, with subsequent methylation to MMA^V, which is then reduced to monomethylarsonous acid (MMA^III). MMA^III is oxidatively methylated to DMA^V, which can be reduced to dimethylarsinic acid (DMA^III) (Tsuji et al. 2019; Zhou and Xi 2018). In rodents and humans exposed to extremely high concentrations of arsenic, DMA^III can be further methylated to trimethyl arsenic oxide (TMA^VO) and, in rodents, it can be subsequently reduced to volatile trimethylarsine (TMA^III) (Cohen et al. 2013). The reduction reactions are catalyzed by glutathione-S-transferase omega (GSTO); and oxidative methylation is mainly performed by the arsenic-3-methyl transferase enzyme (AS3MT) (Naranmandura et al. 2006; Tsuji et al. 2019; Zhou and Xi 2018).

The clinical features of acute arsenic poisoning include, but are not limited to, nausea, vomiting, diarrhea, severe abdomen pain, skin rash, and seizures (Ratnaike 2003). Depending on the amount ingested, arsenic can induce severe systemic toxicity and death. Although acute exposures to high levels of arsenic are occasionally reported, chronic exposure to low levels over a long period of time is more common and currently of great concern. The long-term effects of arsenic exposure differ between individuals, population groups, and geographical areas and clinical outcomes include diabetes, pulmonary and cardiovascular disease, skin lesions, hyperkeratosis, and, as discussed earlier, skin, urinary bladder, and lung cancers (IARC 2012; States 2015; WHO 2018). The mechanisms by which arsenic causes cancer remains elusive, but some of the proposed mechanisms of action are inhibition of DNA repair, oxidative stress, aneuploidy, aberrant DNA methylation, and miRNA dysregulation (Hughes 2002; Tam et al. 2020).

Because of arsenic’s multisystem toxicity and carcinogenic potential, it is important to prevent further exposure to this substance by providing safe water supplies for drinking, cooking,

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