Bioreductive metabolism of AF-2[2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] combined with 2-nitroimidazoles. Implications for use as hypoxic cell markers.

Metabolism of misonidazole under hypoxic conditions depletes the parent drug and causes about 4% of the reduced-drug-products to form adducts with cellular macromolecules (binding), and this process has been used to detect hypoxia in cells and tissues. The nitrofuran, AF-2 [2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] has been shown to increase both the metabolic depletion of misonidazole and its binding. In the present study, factors which might affect this process have been examined, in an in vitro system, to test the hypothesis that metabolic depletion of misonidazole could limit its ability to diffuse freely to the hypoxic cell population. Drastic reductions in glucose concentrations from their normal value of 5-10 mM to less than 0.5 mM had no significant effect on the metabolism of either misonidazole or AF-2. Similarly, glucose concentration did not influence the binding of misonidazole, even when concentrations of both oxygen (extreme hypoxia) and glucose were near zero--a very toxic biochemical environment. Similarly, the metabolism of the nitroheterocyclics had no effect on glucose consumption. The bioreductive depletion of misonidazole in extreme hypoxia appeared to be independent of drug concentration between 25 and 100 microM: this nearly zero-order rate of drug metabolism prevented the possibility of working at constant drug concentration. AF-2 exacerbated this effect by greatly enhancing the metabolic depletion of misonidazole. AF-2 was found to increase both the metabolic depletion and binding of misonidazole by the same factor. An unexpected finding was that metabolism of etanidazole, a 2-nitroimidazole closely related to misonidazole, was not enhanced by AF-2. Micromolar amounts of oxygen inhibited the reductive activation of AF-2, and also the interaction between AF-2 and misonidazole. Our results suggest that metabolic depletion of nitroheterocyclics could influence their ability to diffuse adequately to hypoxic tissues, particularly at the low drug concentrations that have been used to measure tissue hypoxia in vivo.

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse.

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231-246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83-90), which is essential for the biosynthetic activities of the embryo.