Biophysical activity of two twin organometallic compounds Triphenyltin chloride (TPhT) and Triphenyllead chloride (TPhL) in their interreaction with model membranes, as well as with yeast cells
Organometallic compounds, due to their high biological activity, are the object of intensive research conducted by biologists, geneticists, biochemists, and biophysicists. The research focuses mainly on toxic properties of lead, mercury, and cadmium derivatives [
Man [
Four measurement methods were used in the experiments: two physical ones (the spin probes method and the electric method) in studies of the model membranes, and two biological (minimal inhibitory concentration /MIC/ and test of yeast survival) in studies of the
The Egg yolk lecithin (EYL) used in the experiment was prepared at the Institute of Chemistry of Opole University, according to a method described in [
The spin probes: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxy (16-DOXYL-stearic acid methyl ester) as well as the ester of lauric acid were delivered by ALDRICH.
The 50
The liposomes were formed from EYL in distilled water, in the process of sonification. The total sonification time of a single sample (1.5 ml) lasted to 5 minutes, and the sonification was conducted in consecutive cycles consisting of 30 seconds of sonification followed by 30 seconds of cooling. The concentration of the EYL in the sample was 0.04M and that of the spin probe (added to the formed liposomes) was 0.05% in with regard to the lecithin. Two twin compounds, TPhT
The chemical structure of the TPhT and TPhL molecules.
Two spin probes, penetrating different areas of the membrane, were applied in the experiment: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxy (16-DOXYL-stearic acid methyl ester). The TEMPO probe dissolves both in the hydrophobic part of liposome membranes and in water environment, whereas the 16-DOXYL-stearic acid methyl ester locates deep in the middle part of the hydrophobic layer of the membrane.
On the basis of the ESR spectrum of the TEMPO probe, the spectroscopic parameter
ESR spectra of spin probes placed in the membrane of EYL liposome; (a) TEMPO probe, (b) 16-DOXYL- stearic acid methyl ester.
On the basis of the spectrum obtained using the 16-DOXYL-stearic acid methyl ester probe, the spectroscopic parameter
Experimental system consisting of two chambers (the measuring and the reference ones), separated with a membrane (Synpor filter impregnated with lauric acid), was used. The chambers of the measuring system were filled with 10 mM solution of KCl and connected by means of salt bridges with intermediate chambers, in which measuring electrodes Ag/AgCl, connected to the electrometer, were immersed. Such a system prevents an interaction that could occur between the measuring electrode and the compound introduced in the chamber. Using the Keithley 6517 electrometer, the time dependence of the transmembrane voltage (TMV), induced by the presence of admixtures of the examined compounds, was measured. Similarly as in the case of the ESR method, the investigated compounds were dissolved in DMF and added—in the same form—to the measuring chamber. The measurements were carried out for two different concentrations of the admixtures: about 1% and 10% (molar) relative to the lauric acid. The concentrations of the admixtures corresponded, approximately, to those applied in the ESR-based measurements.
Each of the ESR and electric measurements was repeated 5 times, and the presented results are the arithmetic means of the measurements. Relative measurement errors amounted to 1.5% for the parameter
The minimal inhibitory concentration was determined, as previously [
The survival test of the yeast treated with the Triphenyltin chloride and Triphenyllead chloride was investigated in liquid YPD medium. Cultures inoculated with
Figure
Time dependence of the value of the spectroscopic parameter
Figure
Time dependence of the value of the spectroscopic parameter
Analyzing the changes in the ESR spectroscopic parameters of the TEMPO and 16-DOXYL stearic acid methyl ester probes, it may be concluded as the following.
Despite of the similarity of their structures, the tin containing compound—TPhT—interacts with EYL liposome membranes in their hydrophilic and hydrophobic parts more actively than its lead containing twin-TPhL. Its activity increases with concentration.
The changes in the fluidity of membranes, induced by the presence of TPhT in the hydrophobic area, were decisively greater than those in the hydrophilic one, which can testify to a good penetration of the hydrophobic environment by this compound.
Figure
Time dependence of the membrane voltage for (a) 1% concentrations of the TPhT and TPhL; (b) 10% concentration of the TPhT and TPhL admixture in the measurement chamber.
The maxima visible on Figures 5(a), 5(b) testifies to the existence of two competing processes. The first one leads to an increase in the trans-membrane voltage and consists in a gradual penetration particles positively charged of admixture dispersed charge in the electrolyte, into the inside of the membrane. This process is additionally stimulated by the negative charge of the membrane (impregnated filter) applied in the experiment. The other process leads to lowering of the trans-membrane voltage by absorption of Cl- ions by the membrane and begins with a delay in relation to the first process, since it requires neutralization of the negative charge of the filter.
The investigation of the influence of TPhT and TPhL admixtures on model membranes by means of the electric method confirmed the conclusions arrived at with the use of the ESR method: the tin compound (TPhT) displays a greater activity than the lead-containing compound, TPhL, while interacting with lipid membranes. Both methods confirmed that the organic tin chloride more actively interacts with model membranes than the lead chloride, despite the fact that these compounds have identical chemical structures. An analysis of the changes in the TMVs leads to the following conclusions.
The tin containing compound—TPhT—is more active in interactions with membranes of filters impregnated with lauric acid (similarly as in the case of liposome membranes).
The induced TMVs strongly depend on the concentration of investigated compounds, and—in the case of TPhT at the concentration of 10%—it even resulted in a change in the polarization of the system. The compound containing tin, by heavily fluidizing the structure of the membrane, could cause local micropores to appear in the filter impregnated with lauric acid, which in turn could provoke migration of ions through the hydrophobic inside of the filter toward its other side—a change in the polarization of the registered TMV.
Table
The minimal inhibitory concentration (MIC) of Triphenyltin chloride and Triphenyllead chloride for
Compounds | MIC ( |
---|---|
Pb compound | 8 |
Sn compound | 5 |
The obtained results testify to the fact that both compounds display a strong inhibitory activity; still, it is the tin compound that appears to exert a stronger influence: it is enough to use as little as 5
The results of the test conducted on the basis of YPD medium in the presence of Triphenyltin chloride and Triphenyllead chloride are presented in Table
Dependence of survival of
Compounds | Concentration ( | Formations of cell colonies in time | |||
---|---|---|---|---|---|
0 hours | 6 hours | 12 hours | 24 hours | ||
Control | 0 | ||||
Pb | 1 | ||||
compound | 5 | ||||
Sn | 1 | ||||
compound | 5 | — |
Time dependence of the concentration of cell colonies containing admixtures of (a) 1
The inhibitory activity of both compounds increases in dependence on the growth time: the longer the cells are grown in the presence of the particular compound, the fewer of them can survive. This experiment points to strong lethal potential of both compounds and, again, the tin compound appears to be stronger as regards its activity in this respect: at a concentration of 5
All the measuring methods applied in the present study point to a far more considerable activity of the compound containing tin (TPhT) in its interactions with membranes in comparison with that for compound containing lead (TPhL).
The effectivity of the interaction of the compounds with the membranes increases together with time and with the rise in their concentration, which was confirmed both by the ESR method and the test of yeast survival.
The greater activity of TPhT in relation to TPhL is connected with the metal ion inside the molecule, and not with its shape.
The change in the polarization of the registered TMV, induced by the presence of the compound containing tin, can be explained as an effect of strong fluidization of the membrane in the hydrophobic part (results of the ESR method), which could cause local micropores to appear in the filter. This, in turn, induces migration of ions through the hydrophobic inside of the filter to its other side. It is possible that the MIC of 5
The strong influence of the kind of ion enclosed within a large molecule on the biological activity of the latter, when interreacting with both biological and model membranes, seems surprising as well. A similar conclusion can be found in [