Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney

Abstract

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl(2) and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl(2), CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25-10 muM-Cd. Cells exposed to CdCl(2) accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl(2) the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 muM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 muM, therefore, the difference in Cd accumulation between CdCl(2) and CdMt was greater (5-6 times) than at concentrations below 2 muM (1-2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 muM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl(2) was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1-2 muM) to CdCl(2) intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10-20 muM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20-35 muM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 muM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl(2) to cell cultures in a serum-free medium. Whereas CdCl(2) in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 muM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl(2) is associated with a difference in Cd distribution.