Mitochondrial assays determination

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... 

As with the Salmonella reversion assay, this shortterm test is conducted both without (— PMS) and with metabolic activation produced by addition of post-mitochondrial supernatant containing rat liver enzymes ( + PMS). These terms are equivalent to — S9 and + S9 in the Ames reversion assay we use the latter designation for both types of bacterial assays. A more sensitive micro-forward mutation bioassay using this TM677 strain to determine the mutagenicity of indoor air particles, including ETS and wood smoke, is described by Lewtas et al. (1987). 

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

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 traditional way to determine OXPHOS enzyme activities is by spectrophotometry. Several assays have been described for all 5 OXPHOS complexes. The assays are performed in homogenates of tissue samples or cultured cells, in crude mitochondria-enriched 600 g supernatants of tissue/cell homogenates, or in mitochondrial preparations from 14000 g pellets derived from 600 g supernatants. Obviously, the higher the... 

Aliquots of 4 qL of mitochondria were used in each 25 qL assay. This is equivalent to 9 qg of mitochondrial protein containing approx 35 4-0 ng of cytochrome c. The integrity of the enriched mitochondria was routinely assessed by determining the amount of cytochrome c released when mitochondria were incubated under assay conditions without any inducers of cytochrome c release. Typically, the amount of cytochrome c spontaneously released from enriched mitochondria during a 30-min incubation at 30°C was less than 15% of the total cytochrome c. 

Until recently, DNA quantification in forensic analyses has most commonly involved the use of slot-blot methods. In the past few years, the forensics community has shifted focus to the use of quantitative PCR as the preferred method for quantification. Both methods allow for human-specific DNA quantification, although qPCR has significantly lower limits of detection and gives an indication of the PCR-amplifiability of the DNA as well. Recently, multiplex qPCR assays have been developed to simultaneously determine the total amount of genomic and mitochondrial DNA, total genomic and male DNA, or assess the extent of DNA degradation for specific forensic applications. Because of the utility of qPCR to the forensic community, we focus on the development of qPCR on microdevices here. 

Figure 5. The data show how patients with confirmed or suspected defects of the mitochondrial respiratory chain can be separated from patients with confirmed long chain fatty acid oxidation defects (data from Figure 4 plus 8 cases of LCHAD). In each case the release of H O from [9,10- H)myristate, [9,10- H]palmitate and [9,10- H]oleate was determined in parallel with at least 3 unaffected cell lines (not all control data plotted). All determinations were in duplicate and the activities for the individual cell lines are expressed as a proportion of the assay mean.

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. 

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