The interaction of tributyllead with lysosomes from rat liver

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Abstract

The interactions of tributyllead with lysosomes from rat liver have been studied. It results that the organometal compound induces a fast alkalinization in energized lysosomes. The interpretation is that the compound is a potent proton carrier. This function could explain the toxicity, in particular at neurological level of the compound.

Introduction

In the last few years the study of the interactions of trialkyl metals with the biological structures have received more and more attention since these compounds are involved in many problems of environmental concerns [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. Therefore, the behaviour and the toxic effects of trialkylmetal compounds have been largely investigated in animals, to study toxicological effects, and in cells and subcellular structures in order to explain the molecular mechanisms responsible for the effects observed in the whole organism.

The molecular interactions of R3PbCl with mitochondria have been largely studied [1], [2], [3] and a model has been proposed which suggests that these compounds act as Cl/OH exchanger: leadtrialkyls enter the mitochondrial membrane as R3PbCl and are extruded as R3PbOH, thus giving rise to a cyclic mechanism which explains some aspects of the toxicity of these compounds.

Recently, a new model has been proposed to explain the interactions of R3PbCl with mitochondria [6]. The authors propose that the system R3Pb+/R3PbOH is an uncoupler (protons carrier) of oxidative phosphorylation: R3PbCl enter mitochondria as aquo-cations R3Pb+ and are extruded as R3PbOH. The consequent cyclic mechanism gives rise to a transport of protons through the membrane with a consequent collapse of the electrochemical potential.

In order to confirm this mechanism, in this paper we have studied the interactions of Bu3PbCl with lysosomes. Like mitochondria, lysosomes are sensitive to the presence of uncouplers, but, differently from mitochondria, they do not produce ATP, but utilise ATP to pump protons in the inner compartment. As a consequence, in lysosomes the internal pH is acid and the potential is low and positive-inside (in mitochondria the pH is alkaline-inside and the potential is high and negative-inside). Furthermore, lysosomes are less complex systems than mitochondria since the respiratory chain which interferes with trialkyllead compounds and proteic channels pore [12] are absent.

Therefore, lysosomes are a good test to confirm the already proposed uncoupling mechanism.

We have chosen the tributyllead derivative since it is the most toxic for animals and the most destructive for cells [4], [11].

Section snippets

Materials and methods

Lysosomes from rat liver have been prepared following the procedure indicated by Savant et al. [13]. The protein concentration has been determined by the Lowry method [14]. The lysosomes, after the last centrifugation of the preparation, have been resuspended in a medium containing: 0.1 M sucrose, 50 mM K2SO4, 20 mM Hepes pH 7.4, 2.5 mM MgSO4 and 500 μM EGTA and stored at 0°C. The protein concentration of the resuspended lysosomes was 20 mg/ml. The dye acridine orange (AO) 1.0 mM in ethanol was

Results and discussion

In lysosomes, the ATP-dependent proton pump (vacuolar-type H+ATPase) pumps the protons into the matrix. Since the membrane is not permeable to protons, their influx gives rise to acidification and, as consequence, to a positive potential in the internal matrix. The positive-inside potential opposes to a marked acidification since the protons are positively charged. In the presence of Cl ions in the suspending medium, the presence of a positive-inside potential induces the uptake of Cl ions

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