Mitochondrial assays measurement

Aqueous extracts of cat s claw were tested for cytotoxicity in four in vitro bioassays using Chinese hamster ovary cells and bacterial cells (Santa Maria et al., 1997). Concentrations of 10, 20, 30, 40, 50, 75, and 100 mg/mL were used. The neutral protein assay (measures inhibition of cell growth), the total protein content assay, the tetrazolium assay (measures mitochondrial succinic dehydrogenase activity), and the microtox assay (measures inhibition of light output from a luminescent bacterium) showed no evidence of cytotoxicity. 

The cell viability was evaluated by measuring mitochondrial dehydrogenize activity with MTT assay. This assay measures the cell activity, proliferation rate and cell viability. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced to insoluble purple formazan granules by all living, metabolically active cells. The precipitated formazan was dissolved in isopropanol, and the absorbance was read at 595 nm. Absorbance values that are lower than the control cells indicate a reduction in the cell activity and viability. Conversely, a higher absorbance rate indicates an increase in cell viability/ proliferation. 

For a quantitative evaluation of cytotoxicity, the cell viability/proliferation can be determined, for instance, by means of the MTT assay. This colorimetric assay measures the metabolic activity of viable cells, once the dissolved MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) can be converted to a water-insoluble purple formazan by mitochondrial dehydrogenase enzymes of living cells. The number of viable cells correlates to the color intensity determined by photometric measurements after dissolving the formazan. 

Hassoun et al. (1993) examined the effects of various pesticides on lipid peroxidation and DNA single strand breakage in the hepatic cells of female Sprague-Dawley rats. Animals were dosed orally once with endrin at 4.5 mg/kg, lindane at 30 mg/kg, chlordane at 120 mg/kg, or DDT (dichlorodiphenyl trichloro-ethane) at 40 mg/kg, or vehicle only (com oil, control). At 6, 12, and 24 hours post-dosing, 4 animals from each group were sacrificed, their livers removed, and prepared for lipid peroxidation assay. Lipid peroxidation was measured calorimetrically by determining the amount of thiobarbituric acid reactive substances (TBARS) formed. Exposure to endrin resulted in a 14.5% increase in hepatic mitochondrial... 

The activity of complex V (ATP synthase) can be conveniently measured in the reverse direction, ATP hydrolysis with a coupled assay thereafter described [72] (Fig. 3.8.6). The use of oligomycin, a specific inhibitor of the enzyme, allows discrimination of the mitochondrial enzyme from any nonmitochondrial ATPases. 

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

A number of studies have reported that the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) in vitro cytotoxicity assay is a convenient method for assessing ceU viability. The main features found with this assay are its ease of use, accuracy and rapid indication of toxicity. This method is of interest particularly when exposure to unknown chemical substances requires the rapid detection and evaluation of toxic effects. Direct comparisons of the IC50 value determined in the MTS assay and in vivo cytotoxicity showed a significant, direct correlation. The highest correlation was found when the MTS assay was compared with test systems using human cell hnes. The MTS assay is based on the conversion of a tetrazolium salt into a colored, water-soluble formazan product by mitochondrial activity of viable cells at 37 °C. The amount of formazan produced by dehydrogenase enzymes is directly proportional to the number of living cells in culture and can be measured at 492 nm. 

The MTT assay is based on the reduction of the yellow-colored MTT by mitochondrial dehydrogenase of metabolically active cells to a blue formazan that can be measured spectrophotometrically. 

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