Heavy metals, such as cadmium (Cd²⁺) and mercuric (Hg²⁺) chloride, are environmental pollutants and nephrotoxic. They induce reactive oxygen species (ROS) and death of kidney proximal tubule (PT) cells by apoptosis and/or necrosis. We have previously shown that exposure of PT cells to 10 mM Cd²⁺ for 6 h increases ceramide-dependent calpain activation and apoptosis (Am J Physiol Cell Physiol 293:839, 2007). Here we investigated common and singular mechanisms of cell death signaling induced by Cd²⁺ or Hg²⁺. Cell death was quantified by MTT assay or Hoe33342/EtBr nuclear staining. Cd²⁺ (10–50 mM) caused apoptosis at 3–6 h. Using dihydrorhodamine 123, Cd²⁺ (10–100 mM) increased ROS as early as 0.5 h in a time and concentration dependent manner. A maximal increase of ~350% was observed with 50 mM Cd²⁺ after 3 h. This was unaffected by fumonisin B1 (FB1), which inhibits de novo ceramide synthesis, and the calpain inhibitor, PD150606, although Cd²⁺-induced cell death was previously seen to be abolished by these inhibitors, which suggest that ROS elevation is upstream of de novo ceramide synthesis and calpain activation in Cd²⁺-induced cell death of PT cells. ATP production, used to measure mitochondrial function, was also unaffected by 10–50 mM Cd²⁺ for 3–6 h. This suggests that mitochondria are not affected by Cd²⁺ at 3–6 h and ROS formation may be the consequence of depletion of intracellular antioxidants and/or inhibition of free radical-detoxifying enzymes.

Depending on concentration and exposure time, Hg²⁺ can cause apoptotic (5–10 mM for 24 h) or necrotic (50–100 mM for 1–3 h) PT cell death. High Hg2+ (50 mM) induces a ~400% increase in ROS, which peaks at 1 h and returns to control levels, whereas only small increases in ROS were observed with low Hg²⁺. In contrast to Cd²⁺, depletion of ceramide using FB1 augmented ROS production but abolished ATP depletion induced by Hg2+ after 3 h. However, synthetic C6-ceramide did not affect ATP levels, even though cell death by C6-ceramide was evident. The calpain inhibitor PD150606 attenuated Hg²⁺-induced ROS, suggesting involvement of calpains in the propagation of mitochondrial dysfunction. Thus, cell death by Hg²⁺ is governed by the induction of ceramide and early activation of calpains. Contrary to Cd²⁺, ROS formation appears to be a consequence of mitochondrial damage rather than a component of the cell death signaling pathway induced by Hg²⁺.

In conclusion, we demonstrate that although similar components of the cell death signaling pathway are utilized by both Cd²⁺ and Hg²⁺, the sequence of events and consequential effect of each component differs. This may form the basis for differing forms of cell death induced by toxic heavy metals that propagate damage of the kidney PT.