Cytotoxicity quantitative determination

To study the mechanism of cytotoxic action of CNT we used a modified method of spin labels [20], which allows the quantitative determination of CNT influence on membrane integrity of human blood erythrocytes, and mitochondrial activity of hepatocytes in rat liver homogenate, without extraction of mitochondria from cells. 

Uchino T,Tokunaga H, Ando M, Utsumi H. Quantitative determination of OH radical generation and its cytotoxicity induced by Ti02 - UVA treatment. Toxicol in Vitro 2002 16 629-35. 

An LC-MS-MS method for the simultaneous quantitative determination of actinomycin-D (Act-D) and vincristine (VCR), which are cytotoxic agents commonly used in the treatment of pediatric cancers. Following sohd-phase extraction, plasma samples are separated and analyzed using electrospray ionization (ESI). Lower limit of quantitation (LLOQ) for both Act-D and VCR 0.5 ng/ml. Analytical accuracy for detection of both Act-D and VCR <90%. Analytical precision, as estimated hy the coefficient of variation <6% for Act-D and <11% for VCR. Useful in clinical monitoring. 

The cause of the cell cycle specificity of the bisindole alkaloids may be associated with the ability of these compounds to interact with the protein tubulin and thereby inhibit the polymerization (and depolymerization) of microtubules (16,17). In this respect the cellular pharmacology of vinca alkaloids is similar to that of other cytotoxic natural products such as colchicine or podophyllotoxin. On closer inspection, however, Wilson determined that the specific binding site on tubulin occupied by vinblastine or vincristine is chemically distinct from the site occupied by the other natural products (18). Subsequent experiments have determined that the maytansinoids, a class of ansa-macrocycles structurally distinct from the bisindoles, may bind to tubulin at an adjacent (or overlapping) site (19). A partial correlation of the antimitotic activity of these compounds with their tubulin binding properties has been made, but discrepancies in cellular uptake probably preclude any quantitative relationship of these effects (20). 

Little quantitative work has appeared on the determination of the rate of conversion of the various purine analogues to their nucleotides with highly purified phosphoribosyltransferases from mammalian cells or any other source, but there would appear to be a rough correlation between the cytotoxicity of these analogues and their ability to serve as substrates (the Kj values for a number of purines and purine analogues have been determined [45a, 85a] but this value is not a measure of conversion to nucleotide). Table 2.1 lists a number of purine analogues and an estimate of their ability to serve as substrates for the phosphoribosyltransferases. 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

Fatty acid ethyl esters may be clinically important, independent of whether FAEEs induce cytotoxicity, because they can serve as a marker for ethanol intake. We performed a study to determine the clinical utility of FAEE in the blood as a short-term confirmatory marker for ethanol intake and as a long-term marker for ethanol intake after ethanol is no longer detectable (Doyle, 1996), To isolate FAEEs from plasma for quantitation by gas chromatography-mass spectrometry (GC-MS) in these clinical studies, we developed a two-step method using solid-phase extraction with a recovery of 70 3%, using ethyl oleate as a recovery marker (Bernhardt, 1996). 

The numerous methods applied and the end-points measured in cytotoxicity determination can be performed by either qualitative or quantitative means. The following examples and results on the cytotoxicity of metals and implantable alloys correspond to 8.5.1.b Quantitative evaluation of the above-mentioned standards ... 

Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13], a quantitative, convenient method for evaluating a cell population s response to external factors, whether it be an increase in cell growth, no effect, or a decrease in growth due to necrosis or apoptosis. 

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. 

Duarte M et al (2009) A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents. Exp Parasitol 123 195-198... 

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