RESUMEN
Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines did not correlate with GSH levels determined biochemically. Good agreement between the two assays was observed for the rodent lines following depletion of GSH by buthionine sulfoximine, but the level of GSH depletion achieved in the human and monkey lines was always underestimated by MBCl/flow cytometry. These discrepancies were not resolved by increasing stain concentration or staining time. Total glutathione S-transferase (GST) activity and GST isozyme profiles were determined for each of the cell lines. Western analysis with antibodies raised against rat Ya, Yb1, and Yc and human pi isozymes revealed that the rodent cell lines expressed abundant alpha (Ya, Yc subunits) and mu (Yb1 subunits) class isozymes. In contrast, GST-pi was the predominant isozyme detected in the human tumor cell lines and Cos-7 monkey cells. Michaelis-Menten analysis with purified GSTs from rat liver as well as purified human placental (pi) GST revealed that the conjugation of MBCl and GSH catalyzed by the alpha (1-1 and 2-2) and mu (3-3 and 3-4) class GST isozymes was approximately 10 and 80 times more efficient than was conjugation by the GST pi form, respectively. These data indicate that the GST-catalyzed conjugation of GSH and MBCl is isozyme dependent and that MBCl is a relatively poor substrate for the pi isozyme. As a consequence of this isozyme rate differential, the MBCl/flow cytometry technique for GSH quantitation must be applied cautiously, particularly with human tumor cells, many of which have been shown to have high GST-pi activity. Application to other cell types should also be made after careful characterization of GSH levels and GST isozyme composition and only after comparison with other independent assays of GSH concentration.
Asunto(s)
Citometría de Flujo/métodos , Glutatión Transferasa/análisis , Glutatión/análisis , Neoplasias/química , Pirazoles , Animales , Biomarcadores , Humanos , Células Tumorales Cultivadas/químicaRESUMEN
Chemosensitization of bifunctional alkylators by misonidazole (MISO) and related nitroimidazoles in vitro has been shown to require hypoxic exposures. Presumably, reductive metabolism of the nitroimidazole under hypoxic conditions results in generation of a chemosensitizing intermediate(s) in a manner analogous to that described for the hypoxic toxicity of these compounds. In an attempt to identify these intermediates, we examined the ability of reductive metabolites of a model 2-nitroimidazole compound, 1-methyl-2-nitroimidazole (INO2), to enhance the toxicity of melphalan (t-PAM) in HT-29 human colon cancer cells. INO2 was a modest chemosensitizing agent, enhancing L-PAM only under hypoxic conditions. The 2-electron reduction product, 1-methyl-2-nitrosoimidazole (INO), was a potent chemosensitizer, enhancing L-PAM toxicity at micromolar concentrations under either aerobic or hypoxic conditions. In contrast, the 4- and 6-electron reduction products, 1-methyl-2-[hydroxylamino]imidazole and 1-methyl-2-aminoimidazole, respectively, failed to modify cell kill by L-PAM even at millimolar concentration. These results suggest that nitrosoimidazoles may be the active chemosensitizing species generated upon the reductive metabolism of nitroimidazoles.
Asunto(s)
Melfalán/toxicidad , Nitroimidazoles/administración & dosificación , Butionina Sulfoximina , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Glutatión/metabolismo , Humanos , Técnicas In Vitro , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Nitroimidazoles/metabolismo , Oxidación-Reducción , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The biological effects of 1-methyl-2-nitrosoimidazole (INO), the 2 electron reduction product of biologically active 1-methyl-2-nitroimidazole, were examined in HT-29 human colon cancer cells by clonogenic assay and glutathione (GSH) determination. INO was very toxic towards HT-29 cells and was equally toxic under aerobic and hypoxic conditions. Cytotoxicity was highly dependent on cell concentration, decreasing as cell concentration increased. INO also resulted in an initial dose-dependent depletion of intracellular GSH which plateaued when the GSH content of INO-treated cells reached approximately 8% of control levels. As was the case for cytotoxicity, the magnitude of GSH depletion by any given INO dose was highly dependent on cell concentration, being greatest at low cell densities. Both toxicity and GSH depletion were more pronounced when cells were exposed in culture medium without the reducing agent, ascorbate. HT-29 cells preincubated with the GSH synthesis inhibitor, buthionine sulfoximine (BSO), to reduce GSH levels to approximately 10% of control levels were more sensitive to subsequent INO exposure. These data suggest that the nitroso- reduction product of 2-nitroimidazoles may be responsible for cytotoxicity and glutathione depletion associated with hypoxic exposure to 2-nitroimidazoles.
Asunto(s)
Glutatión/análisis , Nitroimidazoles/farmacología , Butionina Sulfoximina , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/análisis , Neoplasias del Colon/patología , Humanos , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have investigated the relationships between nitrosourea structure and physicochemical properties and the ability of misonidazole (MISO) to potentiate nitrosourea cytotoxicity in an in vitro model system. EMT-6/Ro tumour cells were exposed in suspension to each of 9 different nitrosourea anti-tumour drugs under hypoxic and aerobic culture conditions. Additional cultures were similarly treated with nitrosoureas in the presence of 1.0 mM MISO. Seven of the 9 nitrosoureas did not demonstrate any selective toxicity toward aerobic or hypoxic cells. In contrast, chlorozotocin (CHLZ) was slightly more toxic toward hypoxic cells while Bis-OH CyNU more effectively killed aerobic cells. The addition of MISO to the drug treatment enhanced the effectiveness of all the nitrosoureas under hypoxic conditions, with the exception of CHLZ which was uninfluenced by MISO. The magnitude of the MISO dose enhancement factor (DEF, defined as the ratio of drug doses required to reduce cell survival to S = 10(-3) in 4 hours in the absence and presence of 1.0 mM MISO) for each combination was examined as a function of the relative carbamoylating or alkylating activity of the nitrosourea included in that combination. Such an analysis revealed a significant (P less than 0.05) positive correlation between relative carbamoylating potency and DEF. No significant (P greater than 0.20) relationship could be established for DEF and alkylating activity.