Frozen mitochondria as rapid water quality bioassay

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 elec- trode that was interfaced to a PC to collect test analysis data. Blank tests were carried out to verify the oxygen con- sumption linear fitting. Toxicity tests were performed using pure organic and inorganic compounds, such to verify the FM22 sensitivity. A piecewise regression, through an Excelâ Macro, identified the break-point in the oxygen con- sumption and calculated the toxicity. The IC50s 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

AN ADAPTIVE METHOD TO FOCUSING IN AN UNKNOWN SCENARIO

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Frozen mitochondria as rapid water quality bioassay

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 elec- trode that was interfaced to a PC to collect test analysis data. Blank tests were carried out to verify the oxygen con- sumption linear fitting. Toxicity tests were performed using pure organic and inorganic compounds, such to verify the FM22 sensitivity. A piecewise regression, through an Excelâ Macro, identified the break-point in the oxygen con- sumption and calculated the toxicity. The IC50s 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

Improvement on frozen mitochondria bioassay: a methodological remark.

The protocol for the bioassay with the mitochondria of beef heart frozen at -22 °C, (developed by Iero, Manente, Bragadin and Perin, in Chemosphere, 52, 2003) requires that the reaction cell is thermostatically controlled at 25 °C. This value was chosen because it is used as a reference for the state environmental standard (Standard Ambient Temperature and Pressure, SATP). The choice is not, therefore, been supported by assessments on the effectiveness of the test at this temperature, but was dictated by the practice of reporting results to a standard temperature value. Finally, it was decided to make a comparison between the working temperature of 25 °C and the 37 °C one, chosen as close to cattle body temperature (estimated to be 38.6 °C), then "normal" working temperature for mitochondria extracted from heart

VNS in drug resistant epilepsy: preliminary report on a small group of patients

<p>Abstract</p> <p>Background</p> <p>In 1997 Vagus Nerve Stimulation (VNS) received approval from the US Food and Drug Administration (FDA) as an adjunctive therapy in the treatment of medically intractable partial epilepsy in people aged 12 years and older who are ineligible for resective epilepsy surgery. Although the exact mechanisms of action are unknown, the use of VNS with children has increased, including those younger than 12 years of age, or those with generalized epilepsy.</p> <p>Methods</p> <p>We describe the outcome for the first group of nine patients, aged 8-28 years, who had pharmaco-resistant epilepsy and were treated with VNS. During the follow up, we gradually and slowly increased the parameters of the stimulation in order to assess the efficacy of VNS even at parameters which would usually be considered "non-therapeutic", along with possible side effects and changes in quality of life.</p> <p>Results</p> <p>At the last follow, up 1 patient was "seizures free", 3 were "very good responders", 3 were "good responders" and 2 were "non responders". We obtained an initial seizure reduction with low stimulation parameters, the highest current reached being 2.00 mA. This observation supports the possibility that, for younger patients, lower stimulation intensities than those commonly used in clinical practice for adults can be therapeutic. We also wanted to underline the reduction in seizure frequency (~91,7%) and the reduction in seizure duration (> 50%) in the patients affected by drug-resistant absence epilepsy. Adverse effects were mild, tolerable and, in most of cases, easily resolved by adjusting the stimulation parameters. Hoarseness of voice was the most frequent side effect. The improvements in the quality of life are relevant and seem to be independent of the VNS effect in controlling seizures.</p> <p>Conclusions</p> <p>Our small experience seems to confirm the efficacy and safety of VNS in drug resistant partial and generalized epilepsy in developing age groups.</p

A new procedure for the monitoring of Cationic Detergents in solution

The paper describes a new procedure for the selective monitoring of cationic surfactants in solution. The procedure is based on the fact that cationic surfactants are accumulated inside mitochondria by a potential-driven mechanism. Once inside, the surfactant induces the release of the dye Safranine, previously accumulated inside mitochondria. Therefore the monitoring consists of a direct spectrophotometric measure of the rate of release of safranine in the resuspending medium containing the cationic surfactant

A study of the toxic effects of six dibenzofuranes in mitochondria from rat liver

The interactions of five dibenzofurans with the mitochondria from rat liver have been investigated. Results indicate that at low doses all dibenzofurans induce inhibition of ATP synthesis. The efficiency of ATP synthesis is measured from the ratio (R ) between the ADP-stimulated respiratory rate (State 3) and basal respiration (State 4). When R = 1, no ATP synthesis occurs. A comparison between the R values for all investigated dibenzofurans (PCDF) shows that the R values are in the same order of magnitude

FM22 (Frozen Mitochondria bioassay): an animal alternative bioassay for toxicity measures for water soluble samples.

Several methods have been proposed, using responses of whole organisms. The problem, however, is not only strictly scientific, but also involves cost, resources and time. For example, assay with organisms require expensive testing facilities and long operational times are necessary for toxicity measurements. In order to evaluate potential compound toxicity (acute and sub-chronic), we standardized a bioassay using mitochondria of beef hearth, and their applicability and sensitivity was verified. In respect to other based on mitochondria tests, this bioassay (called FM22) showed unquestionable advantages: i) to freeze mitochondria at -22 °C instead of the classical -80 °C, ii) to perform a very big quantity of biological test using always the same mitochondria pool (avoiding differences from age, sex, or health status depending on different organisms); iii) to identify quickly a tested compounds IC50, easily comparable. FM22 end point is the inhibition of mitochondrial respiratory chain and this event is quantified by oxygen monitoring. The oxygen consumption was measured by a Clark electrode that was interfaced to a PC to collect test analysis data (1200 in 20 Macro, identifiedmin run). A piecewise regression, through an Excel the break point in the oxygen consumption and calculated the toxicity. Blank tests were carried out to verify the oxygen consumption linear fitting. Toxicity tests were performed using pure/mix organic and inorganic compounds, elutriates from sea- and fresh-water sediment, sewage, dissolved burned compound sub-products. The FM22 test was a good predictor of toxicity for water and soluble samples; the bioassay is easy, low cost and rapid, then usable for routine tests or like a part of a battery of ecotoxicological tests

An alternative to Neutral Red as a dye for environmental contaminant biomonitoring.

Many methods have been proposed for evaluating of toxic compound presence in water and then to assess the risks to both humans and animals. A common standards procedure for the measuring of contaminant toxicity strength in sea water organisms utilizes the response of a dye (Neutral Red), which accumulates in the acidic vescicles of biological sensors (i.e. Mollusc). When marine Mollusc such as mussels are exposed to pollutants, one of the characteristic pathological alterations is a decreased integrity in the lysosomal membrane. In this work we propose a comparison with the responses obtained with another dye, Acridine Orange. The results show that the response of Acridine Orange is linear in the whole operative pH range (i.e., between pH 4 and 7,4), while Neutral Red is insensitive between pH 6 and 7,4. In addition, Neutral Red shows a protonophore behaviour. We propose therefore that the use of Acridine Orange is preferred to that of Neutral Red, as well that Acridine Orange should be alternative to Neutral Red for the monitoring of stressing environment contaminants by means of biological sensors

Interactions of Cr(VI) and Cr(III) with isolated rat live mitochondria.

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