Toxicity of divalent cations

EDTA is used routinely in biochemistry to chelate divalent metal ions. It was thought this might chelate the calcium and remove it from the system. EDDA and EGTA are also used and are thought to chelate calcium more effectively than EDTA. Neither of these chelators affected the reaction, both were simply shghtly toxic (about 3000 ppm). A series of experiments were run and it was found that foe inhibition of reduction caused by 1.4 to 1.6 pmoles of calcium was relieved by 1 pmoles EDTA. There is not a stoichiometric relationship between EDTA and the metal ion. It is not simply chelating the metal ion. 

It was foimd that 2.5 imoles EDTA would eliminate foe toxicity of all foe ions at their IC50, the concentration of cation that inhibited reduction of the dye 50%. Thus 2.5 pmoles EDTA would eliminate toxicity from 5.5 pmoles calcium but only 0.006pmoles mercmy. It was observed initially that the toxicity of most organic chemicals could be determined in the presence of 2.5 pmoles EDTA. This suggested there could be two mechanisms involved in the reduction of MTT. One is inhibited by toxic organic chemicals and foe second is inhibited by divalent cations. 

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Metal toxicity is also affected by physiochemical factors, such as pH and the concentration of divalent cations. Adding divalent cations, such as zinc, has been reported to mitigate toxicity produced by other metals. For example, the addition of 60 pM zinc reduced toxicity in Pseudomonas putida caused by 3 mM cadmium.148 Zinc had no effect on cells grown in the absence of cadmium. Little is understood surrounding the mechanism of protection however, cadmium uptake was observed to be dependent on zinc concentration.149 Zinc was found to be a competitive inhibitor of cadmium uptake. 

Calcium and magnesium are commonly found in water. Obviously if this assay is to be used with water samples, EDTA must be added. It must be determined using water uncontaminated with organic toxins how much EDTA must be used to compensate for the divalent cations. Often the concentration of divalent cations is determined by atomic absorption spectroscopy. However, these values do not agree with the toxicity relieved by EDTA. Soil samples witii as much as 5 gm calcium (45 mM) per kg soil have been assayed using 2.5 imoles EDTA in each sample (Hillaker, 1996). The calcium is complexed with sulfate and phosphate ions and the calcium is not available to the cell, is not seen by the mechanism that reduces the dye. Levels of soluble calcium and magnesium in water are very low. We have found that 2.5 (imoles of EDTA relieves the inhibition caused by divalent cations in all water and soil samples tested thus far (Botsford, 2000b). 

Table 5.19 lists the 21 QSARs that only used the Pearson and Mawby softness parameter (Op) to predict cation toxicity. Two of these QSARs are duplicates. The Turner et al. (1983) QSAR with r =0.360 is a duplicate of the Williams et al. (1982) QSAR with the same r value. The Turner et al. (1985) QSAR with r =0.879 is a duplicate of the Turner et al. (1983) QSAR with the same r value. The Jones and Vaughn (1978) QSAR for Ag+, Am, Cd " and Hg had the highest perhaps because of their proximity in the periodic table. The Turner et al. (1983) QSAR for Mn +, cm+, Ni +, Cu +, zm+, Cd +, Hg +and Pb " had the second highest r, perhaps because Mn +, Co +, NE+, and Zn " were in row 4 of the periodic table and Cd +, Hg + and Pm+ were in close proximity. For the Jones and Vaughn (1978) and Turner et al. (1983) references with 4 and 3 QSARs, respectively, the r decreased as the number of divalent cations increased (Table 5.18). The Babich et al. (1986) and Magwood and George (1996) QSARs both had high r and almost identical cations. The Babich et al. (1986) QSAR had almost identical cations to those used for the Turner et al. (1983) QSAR with r =0.879 (Table 5.18). The Enache et al. (1999) QSAR also had a high r. However, the Mendes et al. (2010) QSAR with a high r was the best for the highest number (18) of cations (Table 5.19).

The biochemical basis for the toxicity of mercury and mercury compounds results from its ability to form covalent bonds readily with sulfur. Prior to reaction with sulfur, however, the mercury must be metabolized to the divalent cation. When the sulfur is in the form of a sulfhydryl (— SH) group, divalent mercury replaces the hydrogen atom to form mercaptides, X—Hg— SR and Hg(SR)2, where X is an electronegative radical and R is protein (36). Sulfhydryl compounds are called mercaptans because of their ability to capture mercury. Even in low concentrations divalent mercury is capable of inactivating sulfhydryl enzymes and thus causes interference with cellular metaboHsm and function (31—34). Mercury also combines with other ligands of physiological importance such as phosphoryl, carboxyl, amide, and amine groups. It is unclear whether these latter interactions contribute to its toxicity (31,36). 

Some metals can be converted to a less toxic form through enzyme detoxification. The most well-described example of this mechanism is the mercury resistance system, which occurs in S. aureus,43 Bacillus sp.,44 E. coli,45 Streptomyces lividans,46 and Thiobacillus ferrooxidans 47 The mer operon in these bacteria includes two different metal resistance mechanisms.48 MerA employs an enzyme detoxification approach as it encodes a mercury reductase, which converts the divalent mercury cation into elemental mercury 49 Elemental mercury is more stable and less toxic than the divalent cation. Other genes in the operon encode membrane proteins that are involved in the active transport of elemental mercury out of the cell.50 52... 

Disodium EDTA is a special type of molecule known as a chelating agent. EDTA can preferentially bind and sequester divalent cations in the increasing order Ca ", Mg +, Zn ", Pb ". Its role in preservation is to assist the action of thimerosal, BAG, and other agents. By itself, EDTA does not have a highly toxic effect on cells, even in culture. Contact dermatitis is known to occur from EDTA. 

Mechanisms for the toxic effects of inorganic and organic mercury are believed to be similar. It has been suggested that the relative toxicities of the different forms of mercury (e g., metallic, monovalent, and divalent cations and methyl- and phenylmercury compounds) are related, in part, to its differential accumulation in sensitive tissues. This theory is supported by the observation that mercury rapidly accumulates in the kidneys and specific areas of the central nervous system (Rothstein and Hayes 1960 Somjenetal. 1973). 

Many environmentally important chemicals are transported as complexes in natural waters. Complexes may increase or decrease the toxicity and/or bioavailability of elements. Complexation increases the solubility of minerals and may increase or decrease the adsorption of elements. The major monovalent and divalent cations and anions (especially > 10 m) form outer-sphere complexes or ion pairs, in which the bonding is chiefly long-range and electrostatic. Ion pairs are unimportant in dilute fresh waters, but become important in saline waters such as seawater. Minor and trace ions such as Cu, Fe +, Pb +, and Hg are usually complexed, and occur in inner-sphere complexes, which are usually much stronger complexes than the ion pairs. Written in terms of Gibbs free energy. 

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