Mammalian mitochondria asin vitro monitors of water quality

Blondin, George A.; Knobeloch, Lynda; Read, Harry W.; Harkin, John M.

doi:10.1007/bf01606616

Cited by 65 publications

(41 citation statements)

References 8 publications

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“…6,7,10 Also in this case, it is to exclude that such categories of organic contaminants can interfere with the Rotenone inhibition, since generally the concentrations of these compounds do not exceed the micromolecular dose. Rotenone, like NADH, is not removed by BSA because of the high affinity of the compound for the binding site.…”

Section: +mentioning

confidence: 99%

Beef heart mitochondria for the Rotenone monitoring

Mane

Manente

Iero³

et al. 2010

Anal. Methods

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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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Section: +mentioning

confidence: 99%

Beef heart mitochondria for the Rotenone monitoring

Mane

Manente

Iero³

et al. 2010

Anal. Methods

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“…The mitochondrial fraction is a good model for the study of cell toxicity of xenobiotics in energy metabolism, since data from mitochondrial studies are reasonably correlated with cytotoxicity parameters reported for cell cultures and whole organisms [1][2][3]. These organelles provide the eukaryotic cell of nonphotosynthetic tissue with more than 90% of its total energy requirements.…”

Section: Introductionmentioning

confidence: 97%

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

Peixoto

Vicente

Madeira

2003

J Biochem & Molecular Tox

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The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 mol/mg mitochondrial protein) collapses, almost completely, ∆ψ; similar concentrations of 2-chlorobenzoic acid promote ∆ψ drops of about 50%. Proton permeabilization partially contributes to ∆ψ collapse since swelling in Kacetate medium is stimulated, with dicamba promoting a stronger stimulation. The ∆ψ decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the ∆ψ decrease and the change on the O 2 consumption on state 4 respiration; on the contrary, for concentrations above 8 mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.

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“…Thus, more rapid and less expensive biosensors have been proposed (Bulich, 1979;Blondin et al, 1985Blondin et al, , 1987Yamano and Morita, 1993;Argese et al, 1995). Some biosensors use the responses of whole cells and of mitochondria (Knobeloch et al, 1990;Bragadin et al, 1991;Betterman et al, 1996;Riisberg et al, 1996;Palmeira and Madeira, 1997;Wenzel et al, 1997;Manente et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Frozen mitochondria as rapid water quality bioassay

Iero¹,

Manente²,

Perin³

et al. 2003

Chemosphere

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A rapid and relatively low cost bioassay, usable in routine screening water test has been developed modifying the beef heart mitochondria test. In our experiments, mitochondria (FM22) were frozen at )22°C, instead of )80°C (FM80), and their applicability and sensitivity was verified. The oxygen consumption was measured by a Clark electrode that was interfaced to a PC to collect test analysis data. Blank tests were carried out to verify the oxygen consumption linear fitting. Toxicity tests were performed using pure organic and inorganic compounds, such to verify the FM22 sensitivity. A piecewise regression, through an Excel â Macro, identified the break-point in the oxygen consumption and calculated the toxicity. The IC 50 s of the tested compounds were calculated and ranged from 0.123 to 0.173 mg/l for heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) and from 0.572 to 10.545 mg/l for organics (benzene, DMSO, DDE, endrin, dichloromethane, chlorobenzene, 1,2-dichlorobenzene and 1,3-dichlorobenzene). Water effluent samples were then tested. The FM22 gave different toxic reactions to them. Water samples were characterised for heavy metals. The FM22 bioassay had a higher sensitivity than the FM80 and a high reproducibility in the toxicity test with pure compounds. The FM22 test was a good predictor of toxicity for water samples; the bioassay is easy, low cost and rapid, then usable for routine tests.

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Mammalian mitochondria asin vitro monitors of water quality

Cited by 65 publications

References 8 publications

Beef heart mitochondria for the Rotenone monitoring

Beef heart mitochondria for the Rotenone monitoring

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

Frozen mitochondria as rapid water quality bioassay

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