The mechanism of interaction of Cr(VI) with isolated rat liver mitochondria was investigated in this study. The results suggest that Cr(VI) induces the opening of the membrane permeability transition pore (MPT). The phenomenon is cyclosporine-sensitive and is in agreement with the cyclosporine-sensitive apoptosis observed in the cells incubated with this compound. Moreover the action of Cr(III), that is formed in the cells by a reduction of Cr(VI), has been also analysed. The results obtained demonstrated that the Cr(III) does not induce the opening of the MPT in isolated mitochondria, but it has a protective effect in preventing Cr(VI) MPT opening. Therefore, these results suggest that apoptosis is regulated by a balance between Cr(VI) accumulation in the cytoplasm and Cr(III) formation.Many "in vitro" studies regarding the interactions of Cr(VI) with cellular and sub-cellular structures have been performed directed to discover the biological-molecular mechanism responsible for the high toxicity of this element [1-10].Among the numerous cellular investigations carried out, two significant findings stand out:-Cr(VI) enters into the cell (by means of a carrier-mediated mechanism). Cell apoptosis is subsequently induced and the phenomenon is inhibited by cyclosporine A [1,4,6,9,39,41]; -Once inside the cell, the Cr(VI) is reduced to Cr(III) [2,3,5,8].However, these experiments do not clarify whether the apoptosis is induced by Cr(VI) or by Cr(III) since both compounds are present in the cell. In order to clarify this point, the interactions of both Cr(VI) and Cr(III) with isolated mitochondria were investigated in the present study since mitochondria are involved in all apoptosis mechanisms, and the cyclosporine-sensitive cell apoptosis is correlated with the opening of a mitochondrial pore (a MPT cyclosporinesensitive pore) [11,12].The interactions of chromium as Cr(VI) with isolated plants and animal mitochondria were previously studied [7,10], but the conclusions did not clarify the above cited problem. In particular, the opening of the membrane pore and the cyclosporine-sensitivity of this phenomenon, which is the key experiment for determining the involvement of mitochondria, has never been taken into account [7,10]. This study intends to demonstrate that only Cr(VI) form induces the opening of the mitochondrial cyclosporine-sensitive pore, while Cr(III) has no effect on this process, but, conversely, it seems to have a protective effect against the opening of the pore induced by Cr (VI).In mitochondria, the free energy arising from the oxidation of the substrates by molecular oxygen is utilized to form ATP from ADP. The oxidation of the substrates leads to the production of NADH and FADH 2 which triggers the electron flux along the mitochondrial respiratory chain (RC). The electron flow in the RC gives rise to a proton extrusion, that in turn gives rise to a ΔΨ (electrical gradient) and to a ΔpH (chemical gradient) which constitute the proton-motive force (p.m.f. = ΔΨ + ΔpH), driving the ATP synthesis [15]. T...

A new procedure for the selective monitoring of the Rotenone is proposed. Since the Rotenone inhibits the first site of the mitochondrial respiratory chain in all living organisms, the proposed method is based on measurements of inhibition of the respiratory rate of beef heart mitochondria.Since its discovery, Rotenone has been used as insecticide, as fish poison to manage fish populations in reservoirs, lakes and streams, and as eliminating agent of undesirable species. This utilization is due to the toxicity of the compound which inhibits the first phosphorylating site in the mitochondrial respiratory chain. Since Rotenone has been largely used 1,2 and is persistent, it can be an environmental problem.As in the case of many other environmental contaminants, such as phenols, detergents, alkyl-metals and chloroanilines, the availability of biosensors for the monitoring, (in addition to the usual analytical technologies) is very convenient 3-10 since a biosensor warranties low cost rapid measurements, and good reproducibility.Since the action mechanism of Rotenone, which consists in an inhibition of the mitochondrial respiratory chain localized between NAD and the flavoprotein, 11 is the same for all respiratory chains of all living organisms, we propose the utilization of the mitochondria from beef heart as biosensor for the selective monitoring of this compound. The mitochondria from beef hearth have been chosen because they are easily available from animals without any stabular. Moreover, the prepared mitochondria can be stored in freezer for months and can be utilized (in small doses) when necessary. After freezing, the mitochondria are generally no longer able to synthesize ATP, but only in the case of mitochondria from beef heart, the respiratory rate, which is sensitive to the presence of Rotenone, is not affected by freezing, so that they can be stored without losing efficiency.All reagents for molecular biology were purchased from SigmaAldrich (Milan, Italy). MilliQ water was used.The beef heart mitochondria were prepared following the procedure of Azzone et al. 12Once prepared, the mitochondria, subdivided in small amounts (200 ml), were stored in freezer at À20 C and thawed before the measurements.Mitochondrial protein concentration was determined by the Lowry method. 13The respiratory rate (the rate of oxygen consumption) was followed by means of a YSI 5331 Clark oxygen, a selective electrode in a thermostatted (20 C) vessel (2 ml). The 2.5 ml Pyrex vessel, closed by a Teflon cap, was thermostatted at 20 C; the solution was magnetically stirred. Appropriate amounts of sucrose, Mg 2+ , buffer and mitochondria from a mother solution were added to the samples of water containing the Rotenone, in order to satisfy the standard operative conditions.The inhibition of the respiratory chain (RC) by Rotenone takes place at the first phosphorylating site (Fig. 1).The respiratory rate is the rate of oxygen consumption when a reducing substrate is added to the mitochondria. Fig. 2 shows a typical experiment when N...

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