Effect of cadmium on in organello mitochondrial DNA, RNA and protein
--Yu. M. Konstantinov, G. N. Lucenko, V. A. Podsosonny and A. S. Mashnenkov
Studies on molecular biology of mitochondrial DNA (mtDNA) may elucidate the role of this organellar genome in the whole genetic system of the maize cell. Increasing concentrations of Cd in the industrial environment have drawn attention to the toxic effect of this heavy metal ion on properties of the cell genetic apparatus. Little is known about the influence of Cd on the genetic system of mitochondria. In this connection it appears reasonable to examine the effects of various concentrations of Cd on activities of DNA, RNA and protein synthesis in isolated mitochondria.
The mitochondria were isolated from 3-day-old etiolated maize seedlings (hybrid Krasnodarsky 362 ATV) by a standard method of differential centrifugation. Mitochondrial protein was determined by the Lowry method. The DNA was synthesized in mitochondria according to the method of Schegget and Borst (Biochim. Biophys. Acta 95:235-248, 1971) with the use of 32P-dATP (specific radioactivity is >111 PBq€mol-1). The conditions of the RNA synthesis in mitochondria were essentially similar to those described elsewhere (Carlson, J et al., Curr. Genet. 11:151, 1986). The kinetics of the mtRNA synthesis was registered by 32P-UTP (specific activity is >74 PBq€mol-1. The protein synthesis in mitochondria was registered with 3H-labelled amino acids (Forde et al., PNAS 75:3841-3848, 1978). Cadmium was added as CdCl2 to concentrations of 10 to 250µM. The kinetic data were obtained from at least 3-4 experiments.
In the course of our investigations on the effect of cadmium on in organello mitochondrial DNA synthesis, we observed that Cd concentrations of 10 to 150µM had a slight stimulatory effect on this kind of mitochondrial genome activity, while 250µM resulted in a significant decrease of 32P-dTTP incorporation in the mtDNA (Fig. 1).
Figures 2 and 3 show the influence of various Cd concentrations on the activities of RNA and protein synthesis in isolated mitochondria. All Cd concentrations concerned of 10µM to 250µM proved to be inhibitory in a dose-dependent manner.
Page et al. (J. Environ. Qual. 1:288-291, 1972) reported the uptake of cadmium from solutions by corn and other crops. Concerning the effect of various concentrations of cadmium on the genetic system of plants cells, Hirt et al. (Planta 179:414-420, 1989) showed that Cd concentrations of 100-150µM stimulated growth of cell cultures of Nicotiana tabacum L., whereas all other concentrations were inhibitory. Interestingly, RNA and protein syntheses were responsible for the revealed stimulation of cell culture growth. Our data suggest that low Cd concentrations of 10µM to 250µM could affect the mitochondrial genetic processes. The Cd concentration of 250µM had the most pronounced inhibitory effect on all three types of mitochondrial macromolecule synthesis. The apparent similarities between the inhibitory effect of Cd on mitochondrial RNA and protein syntheses imply that Cd affects primarily the rate of mtRNA synthesis and hence the effective concentration of genetic templates available for protein synthesis. The data obtained illustrate high sensitivity of the mtDNA to the deleterious effect of this heavy metal ion. We propose that the mitochondrial genome of corn seedlings is possibly one of the targets of cadmium toxic effects on the plant cell.
Figure 1. Kinetics of mtDNA synthesis in isolated mitochondria in the presence of different concentrations of Cd. 1, control; 2, 10µM Cd; 3, 50µM Cd; 4, 150µM Cd; 5, 250µM Cd.
Figure 2. Kinetics of mtRNA synthesis in isolated mitochondria in the presence of Cd. The designations are the same as in Figure 1.
3. Kinetics of protein synthesis in isolated mitochondria in the presence
of Cd. The designations are the same as in Figure 1.
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