Mining and energy generation, through the burning of coal, are the main anthropogenic activities
in South Africa which leads to heavy metal pollution. Eight metals relevant to metal pollution in
South Africa were identified. These metals are cadmium (Cd), cobalt (Co), chromium (Cr),
copper (Cu), manganese (Mn), nickel (Ni), lead (Pb), and mercury (Hg). In the scientific
literature, the effects and mechanisms of toxicity of single metals and several double metal
combinations are well described. However there is a lack of information on the effects of triple
metal combinations. The aim of this study was to determine the toxic effects of metal
combinations related to direct toxicity and oxidative effects in ex vivo human erythrocytes.
The ex vivo haemolysis assay was used to determine the total toxicity of each metal alone and
in double and triple combinations. It was found that from most to least toxic the metals can be
ordered as follows: Hg >> Co > Cu > Pb > Mn > Cd > Ni. The effect of Cr could not be
determined due to its inherent absorbance at 570 nm. Evaluated at a concentration that caused
10% haemolysis, the combination of Cd:Co:Hg had the highest model deviation ration (MDR) of
2.864 indicating a synergistic toxic effect. An antagonistic effect was observed for several metal
combinations with the strongest effect found, with a MDR of 0.399, for Cd:Cu:Ni.
Toxicity can be a consequence of oxidative damage and therefore the ability of each metal
alone and in double and triple combinations to cause reduced glutathione (GSH) depletion (via
direct binding), catalyse the Fenton reaction (hydroxyl radical averting capacity (HORAC)
assay), malondialdehyde formation (thiobarbituric acid reactive substances (TBARS) formation)
and cause reactive oxygen species (ROS) formation (2 ,7 -Dichlorodihydrofluorescein diacetate
(DCFH-DA) assay) was investigated.
Cu and Hg strongly bound GSH and effectively catalysed the Fenton reaction. For both assays,
observed effects were not synergistic although some metal combinations showed an
Within a cellular environment, MDA formation and reactive oxygen species formation (DCFH-DA
assay) in erythrocytes was the highest for Cu and Hg. Using the DCFH-DA assay a strong
synergistic- and antagonistic effect was observed for the combination Ni:Mn:Hg (MDR = 2.964)
and Co:Cr:Ni (MDR =-0.036) respectively. Regarding MDA formation, the synergistic effect was
the highest for the Cu:Mn:Pb combination (MDR = 4.990) while for the combination Cd:Cr:Pb
(MDR= 0.402) the observed effect was antagonistic.
Reduced glutathione depletion was found to be the highest after exposure to the combination of
Cu:Pb:Hg. The combination of Co:Cu:Hg formed the most hydroxyl radicals according to the
HORAC assay. Lastly the combination of Cu:Mn:Hg caused the most ROS formation of all
combinations according to the DCFH-DA assay.
The metal combinations found in each assay as having one of the highest effect values did
show some correlation between the different assays. Only one of these combinations, the
combination of Cu:Mn:Pb did however have a high reading in four of the five assays used. It
caused more than 15% haemolysis, more than 80% reduced glutathione depletion, high levels
of lipid peroxidation as well as ROS formation.