Differential Metallothionein Isoform Expression as Biomarkers of Cadmium Exposure in Human Urothelium

The metallothionein (MT) superfamily consists of 11 genes whose proteins bind to and sequester metals within cells. Although the presence of so many isoforms raises the possibility of differing metal specificities and functions, they have rarely been discriminated in the literature. This thesis investigated the expression and inducibility of MT isoforms in normal human urothelium with the aim of determining whether specific MT isoforms represent discriminatory biomarkers of cadmium exposure, which has been increasingly linked to the development of urothelial cancer (UC) in recent years. Specialised techniques already established were used for the cell, tissue and organ culture of normal human urothelium. These cultures were exposed to cadmium concentrations reflective of in vivo human exposure and analysed using isoform-specific primers and antibodies. The results revealed the extent of differential expression of the MT isoforms, including preferential metal activation; for example, zinc could highly induce MT-1G transcript expression but could only cause minimal transcript induction of MT-1M. This demonstrates the importance of distinguishing between all known isoforms when determining MT expression. The discriminatory approach used in this thesis allowed the identification of two MT-1 isoforms, MT-1A and MT-1M, whose protein induction was highly specific to cadmium exposure. Although isoform transcript induction was observed to be transient, the protein expression of MT-1A persisted for at least 6 weeks post exposure, consistent with a metal sequestration role. Investigation using spectroscopic techniques additionally suggested that cadmium could penetrate the protective urothelial barrier and enter the underlying urothelial cells, where it may be sequestered by MT within these long-lived cells, thus serving as a long-term source for chronic exposure. Overall the results suggest that MT-1 isoforms may be useful as urothelial biomarkers of cadmium exposure, potentially allowing the identification of individuals ‘at risk’ of developing UC and additionally, stratifying a subset of cadmium-induced UC.