Mitochondrial assays

Ashkenazi, A., and Dixit, V. M. Death receptors Signaling and modulation. Science 281, 1305-1308,1998. 

Caspases The executioners of apoptosis. Biochem. J. 326(Pt 1), 1-16,1997. Fulda, S., and Debatin, K. M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798-4811, 2006. 

Gozuacik, D., and Kimchi, A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891-2906, 2004. 

Hirsch, T., Marchetti, P., Susin, S. A., Dallaporta, B., Zamzami, N., Marzo, I., Geuskens, M., and Kroemer, G. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15, 1573-1581,1997. 

and Krammer, P. H. Death and anti-death Tumour resistance to apoptosis. Nat. Rev. Cancer 2, 277-288, 2002. 

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The toxicologist can still use of in silico technology36 to assess the chemical-based toxicity potential of various structural series and many molecular methodologies can still be applied, including covalent binding assays, mitochondrial assays and oxidative stress assays.29-37... 

Based on the structure of dioxapyrrolomycin and related compounds, it was postulated that the insecticidal activity of these compounds was due to the uncoupling of oxidative phosphorylation. This was subsequently confirmed through mouse-liver mitochondrial assays [6]. 

In 2008, Filipovska and coworkers reported a comparison between cationic and neutral gold(I) complexes as potential anticancer drugs based on the results obtained from the mitochondrial assay [10]. 

Before an antiviral agent becomes a drug, advanced toxicity testing, pharmacological combination, and drug-interaction studies are needed. The use of new cell-based assays that can predict mitochondrial toxicity, lactic acidosis, peripheral neuropathy, anemia, hypersensitivity, lipodystrophy, and other potential side effects can alleviate these issues (Stuyver et al. 2002). 

Intracellular Enzymes Stem Cell Isolation Cellular Membrane Potential Mitochondrial Membrane Potential Gene Reporter Assay Gene Silencing (siRNA)... 

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... 

Assay of Subcellular Fractions for Aldrin Epoxidation. Mitochondrial and microsomal pellets were resuspended in Tris-HCl buffer. Each 5 ml incubation mixture contained the following ... 

Amino-4-cyclopropylidenebutanoic acid (2S)-56, is a methylenecyclopro-pane substituted alanine which can be considered as a non-natural isomer of hypoglycine A 51. It has recently been synthesized racemic [61] and enantiome-rically pure [62]. Biological assays have shown that at relatively high concentration the 5,6-methanoamino acid 56 inhibits the metabolism of pyruvate into glucose, but 56 is not active in inducing the mitochondrial oxidation of fatty acids,Eq. (21) [63]. 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

Dye oxidation (e.g., tetrazolium reductase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt, WST-1 3- (4,5 -carboxymethoxyphenyl) -2-(4-sulfophenyl)-2 H-tetra-zolium, MTS 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, XTT 2,2 -di-p-nitrophenyl-5,5 -diphenyl-3,3 -(3,3 -dimethoxy-4,4 -diphe-nylenej-ditetrazolium chloride, NET), Alamar blue assays, ATP concentration (e.g., luciferase assay), oxygen consumption (e.g., oxygen electrodes, phosphorescent oxygen-sensitive dyes), mitochondrial protein and nucleic acid synthesis mitochondrial mass (e.g., mitotracker dyes) mitochondrial membrane potential (e.g., tetramethylrho-damine methyl ester, TMRM tetramethylrhodamine ethyl ester, TMRE)... 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

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