formazans and Lymphoma

Reactive oxygen species (ROS) are important endogenous etiological agents for DNA damage, and ROS perform critical signaling functions in apoptosis, stress responses and proliferation. The correlation between a lower incidence of cancer in people who consume a diet high in naturally occurring antioxidants and the observed increased ROS in cancerous tissues suggest that antioxidants may be used in cancer chemoprevention. We tested this hypothesis by determining whether the well-described nitroxide antioxidant, tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), acts as a chemopreventative agent in Atm mutant mice, a model of the human cancer prone syndrome ataxia-telangiectasia. Tempol administered continuously via the diet after weaning resulted in an increased lifespan of these mice by prolonging the latency to thymic lymphomas. Tempol treatment reduced ROS, restored mitochondrial membrane potential, reduced tissue oxidative damage and oxidative stress, consistent with antioxidant effects. In addition, this nitroxide lowered weight gain of tumor prone mice without changes in food intake, metabolism or activity level and exhibited an anti-proliferative effect in vitro. Thus, tempol acts as a novel chemopreventative agent in this mouse model of a human cancer prone syndrome, associated with broad antioxidant effects.

Topics: Animals; Antioxidants; Ataxia Telangiectasia; Blotting, Western; Chemoprevention; Cyclic N-Oxides; Formazans; Longevity; Lymphoma; Membrane Potentials; Mice; Mice, Mutant Strains; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Spin Labels; Tetrazolium Salts; Thymus Neoplasms; Weight Gain

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.

Topics: Animals; Cell Line; Dicumarol; Dihydrolipoamide Dehydrogenase; Formazans; Growth Hormone; Humans; Lymphoma; Oxidation-Reduction; Rats; Substrate Specificity; Tetrazolium Salts; Tumor Cells, Cultured

The basic characteristics of MTT-formazan production by both quiescent Nb2 cells and those activated by fetal calf serum or human growth hormone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lactogens such as growth hormone. The resulting in vitro bioassay is sensitive and precise, with a detection limit of about 0.05 mU hGH/l (19 ng/l) and a within-assay imprecision of 2.5% in the presence of 0.3 mU hGH/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, large magnitudes of response are observed. The major component of the response is clearly derived from metabolic activation of the cells, rather than increased cell proliferation. The response was abolished by anti-human growth hormone. Delayed addition of the latter demonstrated that the presence of the hormone is required for the entire 96 h of the recommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plateau which covers 4 orders of magnitude of incremental hormone concentrations. A decline in response is observed at the highest dose tested, 10(6) mU hGH/l (385 mg/l). This auto-inhibition is consistent with recent reports of a reduction in response due to stoichiometric blockade of sequential receptor dimerisation which is crucial for activation of both somatogenic and lactogenic receptors by hGH.

Topics: Animals; Biological Assay; Fetal Blood; Formazans; Growth Hormone; Humans; Lymphoma; Macromolecular Substances; Rats; Staining and Labeling; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Prolactin and other lactogenic hormones are mitogenic for the rat T-cell lymphoma line, Nb2. Glucocorticoids have antiproliferative effects on these cells. A limiting feature of experiments utilizing the Nb2 line is their labor-intensive nature. We therefore adapted the commonly used MTT dye proliferation assay for the Nb2 cell line. While rPRL, hPRL, oPRL, hGH, bPL, and to a lesser extent bPRL stimulated the Nb2 cells, hormones without lactogenic activity, rGH and oGH did not. Human serum and rat sera from animals bearing a PRL-secreting tumor stimulated the Nb2 cells in parallel to standards. Glucocorticoids had anti-proliferative effects on Nb2 cells in the presence of half-maximal or maximal PRL doses, as measured by the MTT proliferation assay. It has been claimed that an oxazoline steroid, deflazacort, has anti-inflammatory effects in clinical studies with fewer of the deleterious side-effects common to glucocorticoids. We therefore compared the in vitro anti-proliferative effects of deflazacort with other glucocorticoids. Deflazacort's negative effect on Nb2 cell proliferation was similar to that of cortisol and prednisolone and less than that of dexamethasone. We conclude that the MTT proliferation assay can be used to study both mitogenic and anti-proliferative substances in Nb2 cells. In addition we found that deflazacort acts similarly in vitro to other glucocorticoids.

Topics: Animals; Anti-Inflammatory Agents; Cell Division; Dexamethasone; Formazans; Glucocorticoids; Growth Hormone; Humans; Lymphoma; Pregnenediones; Prolactin; Rats; Tetrazolium Salts; Tumor Cells, Cultured