Beef heart mitochondria for the Rotenone monitoring

Abstract

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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“…Rotenone is utilized as an insecticide and its toxicity is based on inhibiting mitochondrial respiration 25 . Rotenone is a high affinity mitochondrial NADH dehydrogenase (complex I) inhibitor, which at nM concentrations increases extracellular lactate levels 26 .…”