The role of oxidative stress on the effect of 1,4,7,10,13,16-hexathiacyclooctadecane on copper and zinc toxicity in HepG2 cells.

Experiments have shown that 1,4,7,10,13,16-hexathiacyclooctadecane (L3) increased the Cu2+ toxicity on HepG2 cells, whereas the combination Zn(2+)/L3 was less toxic relative to the metal control. In all cases, glutathione (GSH) levels were decreased and vitamins C and E supplementation partially counteracted the increased toxicity in the Cu(2+)/L3-treated cells. The previously observed effects of this hexathiamacrocyclic ligand (L3) on the Cu2+ and Zn2+ toxicity were further investigated by first depleting the intracellular GSH levels by means of L-buthionine S,R-sulphoximine. Combined treatment with Cu(2+)/L3 resulted in complete cell death, whereas for Zn(2+)/L3 no severe effects were observed. Direct measurement of reactive oxygen species (ROS) revealed that Cu2+ induced a high degree of oxidative stress on the cells. This was not the case for Zn2+. The results proved a previously proposed mechanism in which GSH is used to conjugate the metal-ligand complex, but as a result of this, GSH is no longer available for inactivation of ROS. Also, both the intracellular copper and zinc content were determined for each experiment by means of inductively coupled plasma-atomic emission spectroscopy. According to these data, zinc is depleted in Cu(2+)/L3-treated cells, which could have consequences on superoxide dismutase and as a result of this on the amount of oxidative stress.

The effect of hexaaza- and hexathia-macrocyclic ligands on transition metal cytotoxicity in human hepatoma-derived cultured cells.

The effect of macrocyclic ligands on cytotoxic concentrations of the transition metal ions of copper, zinc, and cadmium was investigated. For this purpose, a hexaaza- [3,6,9,17,20,23-hexaazatricyclo[23.3.1.1(11,15)] triaconta-1(29),11(30),12,14,25,27-hexaene (L2)] and hexathia-chelating ligand [1,4,7,10,13,16-hexathiacyclooctadecane (L3)] were used in the human hepatoma-derived HepG2 cell line. The cytotoxicity was measured by the neutral red uptake inhibition assay. First, the NI50 of the ligands, i.e., the concentration of the ligand inducing a 50% inhibition in neutral red uptake compared to control cells, was determined. In several metal/ligand combination experiments, the effects for L2 were difficult to interpret, whereas for L3 in combination with copper ions, a severe increase -- and for zinc ions, a significant decrease of cell toxicity -- relative to the metal control was observed. To further examine the different effects observed with L3 in combination with, respectively, Cu2+ and Zn2+, the glutathione (GSH) content was measured. The relative GSH content decreased as the concentration of L3 increased. It was proposed that the increased toxicity of the combination Cu(2+)/L3 could be caused by the depletion of GSH and a subsequent inability to scavenge the produced reactive oxygen species (ROS). This hypothesis was supported by experiments during which vitamin E or C was added to the Cu(2+)/L3 system.