Rat basophilic leukemia RBL-2H3 cells show markedly high sensitivity to both CdCl2 and MnCl2 compared with other rat cell lines, due to efficient accumulation of cadmium and manganese. To clarify the roles of metal transporters in hyperaccumulation of cadmium and manganese in RBL-2H3 cells, Cd-resistant and Mn-resistant cells were developed from RBL-2H3 cells by continuous exposure to CdCl2 and MnCl2, respectively. The established Cd-resistant (RBL-Cdr) and Mn-resistant (RBL-Mnr) cells exhibited about 20 times higher LC50 values of CdCl2 and MnCl2, respectively, than parental RBL-2H3 cells, and showed cross-resistance to each metal. The resistance to cadmium and manganese was primarily conferred by a marked decrease in the uptake of both metals. RBL-Cdr cells also showed cross-resistance to HgCl2 and AgNO3 probably due to enhanced expression of metallothionein. Among the possible transporters involved in the uptake of Cd(2+) and Mn(2+), the expression of ZIP8 (Zrt-, Irt-related protein 8), encoded by Slc39a8, showed a marked suppression in both RBL-Cdr and RBL-Mnr cells. These results suggest that ZIP8 plays a pivotal role in the transport and toxicity of Cd(2+) and Mn(2+) in RBL-2H3 cells.

Exposure to excess amounts of manganese causes toxic effects, including neurological symptoms such as Parkinsonism. However, endogenous factors involved in the protection against manganese toxicity remain unclear. Previously, we showed that rat basophilic leukemia RBL-2H3 cells are highly sensitive to MnCl₂ compared with other rat cell lines. To identify the genes involved in resistance to manganese toxicity, two lines of Mn-resistant cells showing resistance to 300 µM MnCl₂ (RBL-Mnr300) and 1200 µM MnCl₂ (RBL-Mnr1200) were developed from RBL-2H3 cells by a stepwise increase in MnCl₂ concentration in the medium. Microarray analyses were carried out to compare gene expression between parental RBL-2H3 cells and RBL-Mnr300 or RBL-Mnr1200 cells. Five genes exhibited more than 10-fold up-regulation in both RBL-Mnr300 and RBL-Mnr1200 cells, and 24 genes exhibited less than 0.1-fold down-regulation in both Mn-resistant cell lines. The S100a9 and S100a10 genes, encoding the calcium-binding S100A9 and S100A10 proteins, respectively, were found among the three most down-regulated genes in both Mn-resistant cell lines. The marked decreases in mRNA levels of S100a9 and S100a10 were confirmed by real-time RT-PCR analyses. Further characterization and comparison of these Mn-resistant cells may enable the identification of novel genes that play important roles in the modification of manganese toxicity.