Robert F. Pittillo

DON, CONV and DONV—III. Pharmacologic and toxicologic studies

Abstract The pharmacologie, toxicologic and oncolytic properties of the ketoamino acids DON ( l -DON; 6-diazo-5-oxo- l -norleucine), CONV ( l -CONV; 5-chloro-4-oxo- l -norvaline; 2-amino-5-chloro-levulinic acid) and DONV ( l -DONV; 5-diazo-4-oxo- l -norvaline) were examined. DON was found to be the most active therapeutic agent of the three drugs against leukemia 1210 and also the most potent cytocidal agent against KB tumor cells in culture. The acute ld 50 values of the agents were dissimilar: CONV was the most toxic drug of the three after single intraperitoneal injections, and DONV the least toxic. Only DON showed evidence of prominent cumulative toxicity. In studies with isolated cells of leukemia 5178Y rendered resistant to l -asparaginase (L5178Y/AR), all three agents appeared to compete both with l -asparagine and with l -glutamine for transport into the cell. DONV competed most effectively with l -glutamine and CONV most effectively with l -asparagine. In mice, all three drugs were cleared from the plasma and excreted into the urine at a rapid rate. None was bound to the proteins of mouse plasma. After an intraperitoneal injection of 100 mg/kg, the concentration of DONV in the pancreas was approximately ten times that of CONV or DON; after comparable intravenous injections, only DONV could be identified in this tissue. Although the metabolism of all three ketoamino acids was found to be minor in degree, evidence is presented that they can be degraded in vitro by organ homogenates and also that purified enzymes can catalyze their transamination. In addition, DON was a good substrate for renal γ-glutamyl transferase (EC 2.3.2.2). In the case of DONV, some conversion to CO 2 by isolated tumor cells also was observed. From these and previous studies it is concluded that, of these analogs of l -glutamine and l -asparagine, DON is the most “ l -glutamine-like” agent of the three, DONV the most “ l -asparagine-like,” while CONV has important attributes of both amino acids.

Screening for and biological characterization of antitumor agents using microorganisms.

Publisher Summary This chapter discusses screening for biological characterization of antitumor agents using microorganisms. The chapter illustrates that great advances have been and are being made (1) in the understanding of some causes of cancer with the promise of being able to reduce cancer incidence by removing or controlling these causative factors and (2) in treatment by surgery and ionizing radiation and supportive post-surgical treatment. Successful biological research efforts demand: (1) inductive reasoning to establish reasonable hypotheses, and (2) usable experimental tools to put the hypotheses to objective test. Searching for anticancer agents in human cancer patients is a practical impossibility for a variety of reasons. Tumor-bearing experimental animals have been the test systems of choice in researches for evaluation of anticancer agents. Screening for effective new anticancer agents and evaluation of congeners of known anticancer agents against a variety of experimental animal tumors demand time, laboratory space, technical labor, money, experimental animals, and the drugs being tested. The use of antitumor-agent-resistant microorganisms as tools for preliminary identification of new anticancer agents and of drug-sensitive or drug-resistant bacteria for gaining information concerning possible sensitive metabolic sites inhibited by new anticancer agents has great potential.

POTENTIATION OF THE LETHAL ACTION OF IONIZING IRRADIATION ON BACTERIA BY CHLORAL HYDRATE AND RELATED COMPOUNDS.

The lethal action of ionizing irradiation on some microorganisms can be enhanced by a number of different materials such as nitrogen mustard (1), hadacidin (2), 2fluoroadenosine (3), bromodeoxyuridene (4), and iodoacetamide (5). In the course of routine screening for radiation modifiers we have observed that chloral hydrate and a number of structurally related materials markedly potentiate the damaging effects of ionizing irradiation on several microorganisms.

Use of Escherichia coli Mutants to Evaluate Purines, Purine Nucleosides, and Analogues

Of 142 purines, purine nucleosides, and analogues tested for inhibition of growth of Escherichia coli B Hill, 45 were active. Of these, 27 were evaluated for inhibition of other E. coli lines, including those resistant to 6-thioguanine, 2-fluoroadenosine, 2,6-diaminopurine, or 6-mercaptopurine. Most toxic to the parent lines were 2-fluoroadenosine, 2-fluoroadenine, 2-fluoro-5′-deoxyadenosine, adenosine, 6-thioguanosine, 6-thioguanine, 6-mercaptopurine, 6-mercaptopurine ribonucleoside, 2-azaadenine, 2′-deoxyinosine, 6-N-aminoadenine, and inosine. Hypoxanthine was strongly inhibitory only to E. coli B Hill. Evidence regarding the substrate specificity of the three purine phosphoribosyltransferases was obtained by assaying for these enzymes in extracts of the various cell lines and by cross-resistance studies. The line selected for resistance to 6-thioguanine had low guanine phosphoribosyltransferase activity (guanosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and was deficient in activity for xanthine and 6-thioguanine. The lines selected for resistance to 2-fluoroadenosine and 2,6-diaminopurine were deficient in adenine phosphoribosyltransferase activity (adenosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), and that selected for resistance to 6-mercaptopurine had low hypoxanthine phosphoribosyltransferase activity and undetectable activity with 6-mercaptopurine as a substrate. Purine, 6-methylpurine, 2-fluoroadenine, 2,6-diaminopurine, and 2-azaadenine were classified as adenine analogues; 6-mercaptopurine and 8-aza-2,6-diaminopurine, as hypoxanthine analogues; and 6-thioguanine and 2-amino-6-chloropurine, as analogues of guanine. The inhibition of bacterial growth by hypoxanthine, inosine, 2′-deoxyinosine, or adenosine was prevented by small amounts of thiamine or by relatively high concentrations of either cytidine or uridine. Cytidine also reversed the inhibition by some purine and purine ribonucleoside analogues. Orotate phosphoribosyltransferase (OMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.10), a possible site of action for these compounds, was not inhibited directly by the toxic agents.

ASCORBIC ACID PROTECTION OF ESCHERICHIA COLI AGAINST RADIATION LETHALITY IN THE PRESENCE OF TETRACYCLINES AND OTHER RADIOSENSITIZERS.

Tetracycline, oxytetracycline, and chlortetracycline present during irradiation have been found to sensitize Escherichia coli ATCC 9637 to the lethal action of ionizing irradiation. Ascorbic acid, which has no significant radiomodifying properties in E. coli, affords marked protection against radiation damage when present in the cell suspensions in the presence of the tetracycline antibiotics during irradiation. The mechanisms by which these compounds exert their radiomodifying effects are not known, although the data suggest possible interference with repair mechanisms.

Cytosine Arabinoside Sensitivity in Actinobolin-Resistant Streptococcus faecalis The Basis of a Utilitarian Microbiological Assay.

Summary The observation that cytosine arabinoside was inhibitory for a strain of Streptococcus faecalis resistant to the antibiotic actinobolin served as the basis for the development of a microbiological assay for cytosine arabinoside in biological materials. Small volumes of less than one ml are sufficient for the assay and as little as 3 μg of cytosine arabinoside/ml of sample can be measured. The biological utility of this assay has been demonstrated.

NEW RADIOMODIFIERS IN BACTERIA.

In the course of routine screening of randomly selected chemicals for radiomodifying activity in microbial systems, a number of compounds have been demonstrated to sensitize bacteria, especially Escherichia coli, to the lethal action of ionizing irradiation (Co60). The compounds reported here are: NSC 45133: Isoindolinium,2-(3-dimethylaminopropyl)-3a,4,7,7atetrahydro-2-methyl-iodide, methiodide CH3 I+ [j [ -CHf-CHi-C CHj-N-CH3 2I CH3

Hadacidin Potentiation of Lethal Action of Ionizing Irradiation. II. Effect on Proliferating Gram-Negative Bacteria.

Summary It may be concluded from this work that hadacidin is a potent potentiator of the lethal effects of ionizing irradiation on proliferating cells of a number of Gram-negative organisms. It appears from preliminary studies that for hadacidin potentiation of ionizing irradiation to be demonstrated, a damaging dose of irradiation must be used and that the compound must be present either prior to or during irradiation. Our data also suggest that hadacidin potentiates irradiation damage regardless of the physiological age of the cells at the time of addition of hadacidin or the amount of time the cells are exposed to it during growth.

A new radioprotector in Escherichia coli.

The procedures used in many of the experiments have been described (2, 3). In essence, the procedure used for measuring effects on nongrowing cells consists in exposing washed cells (grown in a simple glucose-salts medium and resuspended in 0.066 M phosphate buffer) to irradiation from a Co60 source (-1000 Ci, dose rate of 2.0 kR/min), with and without test compound. Experiments with growing cells were performed in which the test compound was added to the glucose-salts medium at the

Biological Assay of Streptonigrin (NSC 45383) in Body Fluids and Tissues of Mice

Streptonigrin, a quinone antitumor antibiotic, has been reported to be effective in human trials. A sensitive and precise microbiological assay for the determination of distribution and concentrations of streptonigrin in the body fluids and tissues of treated mice has been developed in an attempt to supplement successful clinical application of this drug.