and 36 downregulated, while in the serum 46 miRNAs were upregulated and 131 were downregulated. An overlap of 59 abnormally expressed miRNAs was found between serum and urine samples of occupational chronic cadmium poisoning patients; these included miR-122-5p, miR-363-3p, miR-129-5p, miR-204-3p and miR-361-3p (Chen et al. 2021). In individuals exposed to low-levels of cadmium in China, the levels of miR-21 and miR-29b in plasma were upregulated. Serum levels of miR-30 were decreased in Chinese chronic obstructive pulmonary disease (COPD) patients with elevated levels of cadmium in serum by comparison to the levels in healthy control subjects. Additionally, an epithelial-mesenchymal transition was observed in the lung tissue of these patients (Zheng et al. 2021). In another study, individuals in India who were occupationally exposed to cadmium presented elevated levels of serum miR-221 and IL-17, a pro-inflammatory cytokine (Goyal et al. 2021; Zenobia and Hajishengallis 2015). Overall, several studies have identified differentially expressed miRNAs in the urine, serum, and plasma of individuals exposed to cadmium only in the Chinese population.
Circulating miRNAs Associated with Chromium Exposure
Chromium (Cr) is the seventh most abundant element on the earth. As a transition metal, chromium can occur in any of the oxidation states from -II to +VI, but it is mostly found in oxidation states 0, +III, and +VI (Sperling 2005). In the environment, chromium can occur in hexavalent (Cr(VI)) or trivalent (Cr(III)) species (Nath et al. 2009). Cr(VI) has been classified as Group I human carcinogen (Proctor et al. 2014; Straif et al. 2009). Hexavalent chromium is used extensively in industry, for instance in leather tanning, in textile and pigment manufacturing, in stainless steel production, and in welding, polishing, and grinding (Wang et al. 2017). Occupational exposure is thus a major concern, primarily because of inhalation. Once absorbed, chromium circulates in the blood, getting bound to the β-globulin plasma fraction, and is transported to tissues after being complexed with transferrin or other proteins (Pechova and Pavlata 2007). Within cells, hexavalent chromium compounds undergo reduction by ascorbate and non-protein thiols, cysteine, and glutathione, and this leads to the formation of transient and highly reactive Cr(V) and Cr(IV) species, organic radicals, and, finally, Cr(III) products (Manning et al. 1994; Salnikow and Zhitkovich 2008; Wang et al. 2017). Intermediate chromium forms are mostly responsible for oxidative stress and DNA damage, which they do by inducing chromium–DNA adducts, DNA strand breaks, and DNA–protein cross-links, all known to contribute to mutagenesis and carcinogenesis (O’Brien et al. 2003; Wang et al. 2017; Wise et al. 2002).
The acute effects of inhaled hexavalent chromium include shortness of breath, coughing, and gastrointestinal and neurological effects (IARC 1990; USEPA 1998). The chronic inhalation of hexavalent chromium causes ulcerations of the septum, bronchitis, pneumonia, decreased pulmonary function, and increased risk of developing respiratory system cancers, primarily bronchogenic and nasal (IARC 1990; USEPA 1998). The chronic inhalation of high levels of hexavalent chromium induces liver, kidney, and gastrointestinal damage (IARC 1990; USEPA 1998).
Several studies have investigated chromium-induced circulating miRNA dysregulation. miRNA profiles from Chinese workers involved in the production of chromate revealed lower levels of miR-3940-5p associated with high levels of hexavalent chromium in blood (Li et al. 2014). In addition, plasma levels of miR-3940-5p were positively correlated with micronuclei frequency in the hexavalent chromium-exposed group (Li et al. 2014). The micronuclei test has been recognized as a reliable biomarker of genotoxicity in occupationally chromium-exposed individuals (IARC 1990; Sudha et al. 2011). Therefore miR-3940-5p could in principle be a biomarker of chromium exposure linked to genetic damage. A very recent study observed forty-five significant differentially expressed miRNAs in the plasma of hexavalent chromium-exposed workers from China (Jia et al. 2020). RT-qPCR validated both the decreased levels of miR-19a-3p, miR-19b-3p, and miR-142-3p and the increased levels of miR-590-3p and miR-941 in the hexavalent chromium-exposed group (Jia et al. 2020). Expression levels of miR-143 were decreased, whereas Interleukin 6 (IL-6) was increased in the blood samples of hexavalent chromium-exposed workers by comparison with the samples of unexposed workers (Wang et al. 2019). Only one study attempted to better understand the linkage between chromium, miRNAs, and metabolic and cardiovascular diseases. Blood levels of forty-three miRNAs were negatively associated with chromium in the urine of obese subjects in Italy (Dioni et al. 2017). By RT-qPCR, nine miRNAs (miR-451, miR-301, miR-15b, miR-21, miR-26a, miR-362-3p, miR-182, miR-183 and miR-486-3p) were downregulated in association with chromium in urine. miR-451 was further associated with glycated hemoglobin, whereas miR-486-3p was associated with both diastolic and systolic blood pressure in obese subjects who presented urinary chromium (Dioni et al. 2017). These two circulating miRNAs can be considered putative biomarkers of chromium exposure, and their relation with glycated hemoglobin and blood pressure may give insights into chromium-induced metabolic and cardiovascular diseases (Dioni et al. 2017). In summary, studies so far demonstrate a differential expression of several miRNAs upon chromium exposure, albeit with no overlap of a single miRNA across all these studies. This inconsistency is a major caveat, given that all the studies have been conducted on the Chinese population and further substantiation of these findings across other exposed populations is required.
Circulating miRNAs Associated with Mixed Metals Exposure
Often populations are exposed to a mixture of metals released into the environment either through natural processes or as an outcome of anthropogenic activities (Cory-Slechta 2005). While it is important to understand the effect of each metal on human health individually, this does not adequately elucidate the molecular events operative within the environmental reality of mixed metal exposures (Cory-Slechta 2005). This is particularly important, given that exposure to one metal can affect the uptake, metabolism, disposition, and elimination of other metals and their metabolites (Young et al. 2019).
Recently studies have started analyzing how expression of miRNA(s) are altered by exposure to multimetal mixtures. In a study of welders exposed to mixed metal fumes, plasma miR-21 and miR-155 levels were lower than in matched unexposed control populations (Amrani et al. 2020). Furthermore, miR-21 and miR-155 expression levels were demonstrated to be positively associated with urinary chromium levels, while miR-146a was positively associated with urinary nickel levels (Amrani et al. 2020). In another study on sixty-three healthy male steel plant workers occupationally exposed to mixed metal particulate matter, blood leukocyte expression of miR-222 was positively correlated with lead exposure, while that of miR-146a was negatively correlated with lead and cadmium exposures (Bollati et al. 2010). Importantly, only miR-222 was found to be significantly upregulated in workers at the end of the workweek by comparison with the situation at the beginning of the workweek, after two days’ break from exposure to particulate matter. In a subsequent study from the same group, the leukocyte expression of four upregulated miRNAs (miR-421, miR-146a, miR-29a, and let-7g) was found to regulate the downstream expression of eleven target candidate inflammatory genes in a population of ten steel plant workers exposed to mixed metal particulate matter (Motta et al. 2013). In another study, urinary expression of miR-200c and miR-423 was positively associated with chromium concentration in individuals in the upper tertile of urinary chromium levels, but not with arsenic levels in a cohort of Mexican children (Cardenas-Gonzalez et al. 2016). In a study of healthy male coke oven workers in southern China, decreased plasma expression levels of miR-27a-3p were associated with lead exposure (Deng et al. 2019). In another study, on Hong Kong Chinese adolescents, expressions of urinary miR-21 and miR-221 were found to be negatively correlated both with urinary arsenic and lead (Kong et al. 2012).
Multiple heavy metal exposures often interact in a myriad of ways, modulating toxicity and health outcomes (Wah Chu and Chow 2002; Young et al. 2019). No studies thus far have examined whether there is any additive, synergistic, or antagonistic effect between the different metals in terms of miRNA expression modulation. This lack of investigation represents a considerable knowledge gap in the field—and one that needs to be addressed.
Summary of Potential Circulating miRNA Biomarkers for Heavy Metals
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