Increased mutagenicity of chloroethylnitrosoureas in the presence of a rat liver S9 microsome mixture.

The mechanism for an enhanced effect of Aroclor 1254-induced Sprague-Dawley rat liver 9,000 x g supernatant (S9) microsome preparation on the mutagenicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-N-nitrosourea (CCNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose (chlorozotocin) for Salmonella typhimurium strain TA1535 was studied. Although all three compounds were direct-acting mutagens, rat liver S9 increased the mutagenic response to BCNU, CCNU, and chlorozotocin. The enhanced mutagenic effect was independent of NADPH. Heat-denatured S9 enhanced the mutagenicity of BCNU and CCNU, but not that of chlorozotocin. Mutagenic enhancement, however, was less than that observed with untreated S9. The substitution of extractable S9 lipid and bovine serum albumin for S9 in the reaction mixture resulted in an enhanced mutagenicity of CCNU with little or no effect on BCNU or chlorozotocin mutagenicity. These results suggest that the enhanced mutagenicity of CCNU, and possibly that of BCNU, in the presence of S9 was due in part to nonspecific factors that are present in the S9 preparation.

Isolation, synthesis, and antitumor evaluation of spirohydantoin aziridine, a mutagenic metabolite of spirohydantoin mustard.

Spirohydantoin mustard (SHM), a central nervous system directed nitrogen mustard with anticancer activity, was metabolized in the presence of mouse liver postmitochondrial supernatant (9000g fraction) to a nonpolar alkylating metabolite. The metabolite was isolated by thin-layer chromatography of chloroform or ethyl acetate extracts of incubation mixtures, and its structure was established by mass spectral analysis, synthesis, and cochromatography. The metabolite, spirohydantoin aziridine, was mutagenic for Salmonella typhimurium TA1535 in the Ames assay but inactive as an antitumor agent against P388 leukemia in vivo.

Sub-femtomole quantitation of proteins with Threshold, for the biopharmaceutical industry.

The Threshold system provides for rapid quantitation of a variety of analytes at sub-femtomole levels, which is fewer than 6 x 10(8) molecules. The operating principles of the measurement system will be described, including the means for chemical modulation of the signal and the subsequent signal detection utilizing a proprietary silicon sensor. The Immuno-Ligand Assay is a universal ligand-binding assay system which provides quantitative measurement of proteins (including antibodies) in a variety of biological media. Assay performance will be described, along with data demonstrating sensitivity, precision and accuracy, for a variety of analytes of interest to the biopharmaceutical industry.

Diffusion-enhanced energy transfer shows accessibility of ribonucleic acid polymerase inhibitor binding sites.

Rifamycin and Cibacron Blue F3GA are powerful inhibitors of Escherichia coli deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase. In addition, both inhibitors strongly absorb visible light, making them suitable for use as acceptors in energy-transfer experiments. Transfer of energy to these acceptors from freely diffusing energy donors with long excited-state lifetimes (approximately 10(-3) s) depends strongly on whether donor and acceptor can make direct intermolecular contact. We observe that the rate constant for energy transfer from a small terbium chelate to enzyme-bound rifamycin is 1 X 10(7) M-1 s-1, which is about half as large as the rate constant observed for free rifamycin in solution. This relatively small change upon binding indicates that enzyme-bound rifamycin is highly accessible to small molecules in the solvent. In the case of Cibacron Blue, under conditions where approximately 90% of this inhibitor is bound to RNA polymerase, the small amount of unbound inhibitor accounts for practically all of the observed energy transfer. This implies that enzyme-bound Cibacron Blue is relatively inaccessible to energy donors in the solution. The dependence of energy transfer on the accessibility of the acceptor is illustrated by using simple geometric models. synthesis of a stable, electrically neutral terbium chelate which can be efficiently excited with UV radiation is also described.

Radiographic aids in the management of intraocular foreign body in the chamber angle.

A technique of bone free radiography for the purpose of diagnosis of a minute intraocular foreign body and for the purpose of confirming the removal of the foreign body under sterile operating room conditions has been described. In addition to the obvious medical advantage of absolutely confirming the removal of an intraocular foreign body there is also the medico-legal benefit of documentation before leaving the operating room.

Bleomycin, and antitumor antibiotic: improved microbiological assay and tissue distribution studies in normal mice.

A microbiological assay was developed for bleomycin, an antitumor antibiotic reported to be active in human trials. The assay bacterium was a strain of Escherichia coli which is resistant to ethionine. Studies revealed relatively high concentrations of bleomycin in the blood and urine of mice after a single dose, < 0.33 ld(10), injected intraperitoneally.

Laser differential light-scattering bioassay for methotrexate (NSC-740).

A new bioassay method utilizing laser light scattering from suspensions of drug-sensitive bacteria has been developed for the estimation of antitumor drugs in biologic samples. Changes in the light-scattering patterns of antibiotic-treated bacteria have recently been shown to provide a rapid and accurate indication of antibiotic sensitivity. Similar considerations for several antitumor drugs have shown the method capable of assaying 0.1 ml with drug concentrations as low as a few nanograms of drug per milliliter of sample. The first successful application of the methodology is described for the antitumor agent methotrexate. Studies of both drug-treated human serum specimens and dog serum levels and urinary excretion as a function of time indicate that assay results are available within 3 hours of preparing the serum. Time variations of drug serum levels and urinary excretion rates are compared via laser differential light-scattering assay, standard disc-diffusion assay, and previously published radioisotopic assays.