Urine miRNAs as potential biomarkers for systemic reactions induced by exposure to embedded metal

Many veterans live with military grade heavy metal fragments retained in soft tissue. Retained heavy metal fragments may negatively impact health in various organ systems and can manifest as gastrointestinal, neurocognitive, pulmonary and renal disturbances. As such, a better understanding of the long-term effects of retained metals and identification of biomarkers indicative of detrimental health outcomes would benefit clinical decision making. In this study, we analyzed urine microRNAs from rats with military-relevant pure metals implanted in the gastrocnemius muscle for 1, 3, 6, and 12 months. Our results provide potential tissue targets affected by metal exposure and a list of unique or common urine microRNA biomarkers indicative of exposure to one or more metals, highlighting a complex systemic response. Three month old, male, Sprague-Dawley rats (n=8/group) were surgically implanted with one of eight metals of interest (W, Ni, Co, Fe, Cu, Al, Pb, and DU) or tantalum (Ta); Ta, serves as a surgical procedure control because it has been shown to be completely inert when embedded in skeletal muscle. Urine was collected from each rat using hydrophobic sand, one week before and 1, 3, 6, and 12 months after surgically implanting two metal pellets (1 mm diameter x 2 mm length) into each gastrocnemius muscle. Urine collection lasted two hours per session and if the rat produced little or no urine during a session, collection was repeated the next day and urine samples were combined for the two sessions. RNA sequencing was performed on urine samples from two pre-surgery animals per metal and from three-eight post-surgery animals per time-point per metal depending on limited sample volumes available for RNA isolation, totaling 178 samples.