Search PubMed⌕ Search

Biomedical subjects

A M Rauth

Publications and source records attributed to A M Rauth.

At least 73 records · Page 4Linked to original sources
PubMed1986

Stable reduction product of misonidazole.

The predominant stable product (greater than 80%) of the anaerobic radiation chemical reduction (pH 7, formate, N2O) of misonidazole (MISO) has been identified as the cyclic guanidinium ion MISO-DDI, a 4,5-dihydro-4,5-dihydroxyimidazolium ion. This cation was prepared as its sulfate salt by the reaction of glyoxal and the appropriate N-substituted guanidinium sulfate. Its formation during MISO reduction was established by NMR spectral comparison and by derivatization as glyoxal bis-oxime, which was formed in 86% yield in fully reduced systems. The toxicity of pure MISO-DDI X sulfate was examined in vivo (C3H mice) and in vitro (CHO cells). This product is less toxic than the parent MISO and free glyoxal. A reactive, short-lived, intermediate is suggested as the agent responsible for the toxicity of MISO under hypoxic conditions.

Animals↗
PubMed1986

Modification of the cytotoxic activity of mitomycin C by oxygen and ascorbic acid in Chinese hamster ovary cells and a repair-deficient mutant.

The cellular and molecular damage produced by mitomycin C (MMC) in Chinese hamster ovary cells, AA8-4, and a repair deficient mutant of this line, UV-20, was studied by utilizing a system in which oxygen levels could be altered and monitored in solution during acute drug exposures. The cytotoxic activity of MMC decreased from hypoxic conditions to 1% oxygen in solution, while from 1 to 20% there was little change. The relative level of DNA cross-linking in cells was examined under these conditions by alkaline elution and found to increase as cell survival decreased. Utilizing a cell-free assay which detects formation of alkylating species it was confirmed that, while alkylation was observed under aerobic conditions, overall levels increased in the absence of oxygen. The presence of ascorbic acid in the exposure medium (0.284 mM) increased the aerobic but not the hypoxic cytotoxicity of MMC. This resulted in a diminished differential toxicity for cells exposed under aerobic versus hypoxic conditions in the presence of ascorbic acid. When ascorbic acid was present, net alkylation increased under aerobic conditions but was unchanged under hypoxic conditions. One interpretation of these results is that at least two mechanisms of activation of MMC to toxic intermediates may be present in these cells.

Alkylation↗
PubMed1986

Measurement of low levels of oxygen and their effect on respiration in cell suspensions maintained in an open system.

The presence of low levels of oxygen may have profound effects on the cytotoxic activity of radiation, radiosensitizers, and bioreductive alkylating agents. As others have shown, low oxygen tensions may significantly alter rates of cellular and chemical oxygen consumption. When experiments are performed at very low oxygen concentrations, the opposing effects of oxygen leakage into and cellular/chemical oxygen consumption from the system can lead to unpredictable results. Use of a newly designed, highly sensitive Clark-type oxygen sensor has permitted accurate and reproducible measurement of low levels of oxygen. Cellular depletion of oxygen at various cell densities has been monitored for a series of oxygen tensions in solution and the corresponding respiration rates have been calculated. Although oxygen depletion was found to be quite significant at low oxygen tensions, not all oxygen present could be removed by cellular respiration. Respiration rate decreased as oxygen tension decreased and approached zero at low oxygen tensions. This result was independent of cell density. A model is presented to account for the observed effect of oxygen tension on cellular oxygen utilization.

Animals↗
PubMed1985

A bioassay to measure cytotoxicity of plasma from patients treated with mitomycin C.

The unpredictable clinical toxicity observed in patients treated with mitomycin C and the observation that this agent must be reduced to an active form before alkylating target molecules have led to the development of a bioassay which is capable of detecting biologically active forms of mitomycin C in the plasma of drug-treated patients. The bioassay makes use of a repair-deficient mutant of Chinese hamster ovary cells, UV-20, which is 40 to 60 times more sensitive to mitomycin C than its wild-type parent. A standard curve relating in vitro cell colony-forming ability of UV-20 versus drug concentration in the plating medium has been determined. Mitomycin C levels in patient plasma as low as 1 ng/ml can be detected, compared to the 5-ng/ml limit of detection obtained with a high-pressure liquid chromatography assay for the parent compound. This assay has been utilized to detect active drug in plasma obtained from patients with colorectal cancer treated with mitomycin C as a single agent. At the completion of drug injection, serial blood samples were collected in heparinized tubes, and aliquots of plasma were extracted and assayed for mitomycin C levels by high-pressure liquid chromatography, diluted and assayed directly for their toxicity for UV-20 cells, or frozen at -20 degrees C to be assayed at a later time. The activity detected by immediate bioassay was stable up to 2 mo in frozen samples. Plasma pharmacokinetics determined by the bioassay in seven patients were no different than those determined by high-pressure liquid chromatography. No stable, cytotoxic species other than the parent compound were detected by the bioassay in the plasma of patients treated with mitomycin C.

Animals↗
PubMed1984

2-Hydroxylaminoimidazoles--unstable intermediates in the reduction of 2-nitroimidazoles.

An unstable 2-hydroxylaminoimidazole (2-hydroxylamino-1-methylimidazole) was prepared by the reaction of 2-fluoro-1-methylimidazole with hydroxylamine. This substance was sufficiently stable (half-life of 1-2 days) in acid solutions to be observed and characterized by NMR spectroscopy; decomposition at neutrality was, however, rapid (half-life of 1-10 min). Radiochemical and electrochemical reduction experiments were carried out at pH 4 and pH 7 with 2-nitro-1-methylimidazole and misonidazole [1-(3'-methoxy-2'-hydroxypropyl)-2-nitroimidazole]. A four electron stoichiometry was found in every case. The pH 4 reduced product was identified as the 2-hydroxylamino derivative (greater than 80% yield). The pH 7 reduced solutions, on the other hand, showed no aromatic 1H NMR signals, suggesting that a simple imidazole ring was no longer present. A shift to pH 7 of the hydroxylamine produced at pH 4, however, resulted in very similar NMR spectra. The conclusion, therefore, is that the hydroxylamine was produced initially on reduction of the nitroimidazole, but it was not stable.

Chemical Phenomena↗
PubMed1984

Pharmacology and toxicology of sensitizers: mechanism studies.

Nitroimidazoles are being studied extensively as hypoxic cell radiosensitizers. Besides their ability to selectively sensitize hypoxic cells to radiation, which depends on the parent compound, nitroimidazoles have a variety of other effects in vitro, in vivo and clinically which appear to require reductive metabolism. These other effects include direct cytotoxicity to hypoxic cells, mutagenicity and antimicrobial effects. As a first step to suggesting possible mechanisms for these other biological effects, a summary has been made of the known oxidative and reductive products of the two most widely studied radiosensitizers, metronidazole and misonidazole. Focussing on reductive products, it is clear that a great variety exists which are or may be reactive with biological molecules. Knowledge about the reduction chemistry of nitroimidazoles is new and far from complete. As a second step to suggesting possible mechanisms for these biological effects, it is important to view the problem in terms of the in vivo situation where distribution and sites of metabolism of the drug and its reduction products will be important factors. Variables such as levels of tissue oxygenation and nitroreductase activity will be important to assess. Combining basic information about the reduction chemistry of nitroimidazoles with knowledge about the pharmacology of drugs and their reduced products should allow a better assessment of mechanism of action as well as a better implementation of these drugs clinically.

Animals↗
PubMed1984

The oxygen dependence of the reduction of nitroimidazoles in a radiolytic model system.

Radiation chemical reductions using eaq- and CO2- have been carried out in the presence of oxygen with metronidazole, p-nitroacetophenone, misonidazole and three other 2-nitroimidazoles. Low concentrations of oxygen were found to effectively inhibit the reduction of the first two compounds while much higher concentrations of oxygen were required for all of the 2-nitroimidazoles. These results parallel in vitro and in vivo experiments with metronidazole and misonidazole which also indicate that the reduction of the latter is significantly less inhibited by oxygen. Kinetic modelling of the radiochemical system suggests that the explanation for the differences lies in different reactions of the nitro radical anions; it appears that the anion derived from metronidazole undergoes disproportionation while that derived from misonidazole undergoes a unimolecular decay.

Acetophenones↗
PubMed1983

Failure of 2-deoxy-D-glucose and 5-thio-D-glucose to kill hypoxic cells of two murine tumors.

We have administered the glycolysis inhibitors 2-deoxy-D-glucose and 5-thio-D-glucose to C3H/HeJ mice bearing KHT or 16/C transplantable tumors to seek evidence for hypoxic cell toxicity in vivo. The drugs were given (a) with or without insulin, (b) as large single doses or as multiple hourly injections, and (c) alone or immediately after the tumors had received radiation to kill most of the aerobic cell population. Tumor response was assessed by growth delay or by lung colony assay. Limiting toxicity of 2-deoxy-D-glucose and 5-thio-D-glucose was neurological, leading to seizures and/or death, and this toxicity was increased by insulin. The drugs had at most minimal effects on the growth of either untreated or irradiated tumors at maximal tolerated doses. Despite the known selective toxicity of these glucose analogues for hypoxic cells in tissue culture, we have found them to be ineffective in killing hypoxic cells of two murine tumors.

Adenocarcinoma↗
PubMed1983

Activity of mitomycin C for aerobic and hypoxic cells in vitro and in vivo.

We have observed the selective toxicity of mitomycin C toward hypoxic as compared to aerobic cells in vitro for three established cell lines (Chinese hamster ovary, Chinese hamster V-79, and human HeLa) and for cells from the transplantable KHT murine tumor. The magnitude of the selective toxicity was cell line dependent. We have studied the in vivo effects of mitomycin C against aerobic and hypoxic cells of two transplantable murine tumors: the KHT fibrosarcoma and the 16/C mammary carcinoma. Either mitomycin C was given with radiation to kill most of the aerobic cells, or it was given alone. Endpoints of response were cell survival assessed by lung colony assay for the KHT tumor, and growth delay for the 16/C tumor. In some experiments, mitomycin C appeared more effective when used with radiation than when used alone, but the results of combined treatment fell just within the range of additivity as defined by isobologram analysis. The effects of combined treatment were independent of the order in which drug and radiation were given. Mitomycin C was also used in combination with Adriamycin to treat the 16/C tumor, since we have found previously that Adriamycin spares hypoxic cells in this tumor. In three of four experiments, combined drug effects were slightly greater than predicted by an additive relationship. We conclude that mitomycin C is active against hypoxic cells in two murine tumors, but that it has at most minor specificity for hypoxic as compared to aerobic cells in vivo.

Aerobiosis↗
PubMed1982

A phase I study of misonidazole and pelvic irradiation in patients with carcinoma of cervix.

A Phase I study of oral daily misonidazole (MISO) with conventional pelvic irradiation, has been conducted in patients with carcinoma of the cervix Stages IB, IIB, IIIB and IVA. MISO was administered in daily dosages to sequential groups of patients at doses of 0.15 g/m2, 0.30 g/m2 or 0.45 g/m2 for 22 days over 5 weeks. Sixteen patients were assigned to each dose level. Using a double-blind randomization, they received either placebo (3/16) or MISO (13/16). The major dose-limiting toxicity was peripheral neuropathy (PN). None of the 13 patients receiving 0.15 g/m2 or the 13 receiving 0.3 g/m2 developed PN. However, 6/13 at the 0.45 g/m2 level (total dose less than or equal to 9.9 g/m2) developed PN. Additional patients were entered at this level and a total of 13/26 developed PN, which was considered of clinically significant severity in 9. Symptoms of PN have persisted from 1 week to 10 months, and have been completely reversed in 9/13 patients. Pharmacological parameters were examined for correlation with clinically evident toxicities. Although peak plasma MISO levels and half-lives did not correlate significantly with PN, there was a significant correlation between the calculated "area under the curve" (AUC) and PN. No correlation exists between PN and total urinary excretion of MISO or the O-demethylation product. A daily dose of 0.45 g/m2; MISO (total dose less than or equal to 9.9 g/m2) is considered to produce an acceptable level of toxicity for this patient population.

Double-Blind Method↗
PubMed1981

Selective toxicity of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide toward hypoxic mammalian cells.

The chemotherapeutic agent 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) is used in the treatment of malignant melanoma where response rates of 15 to 30% have been reported. Some current interest exists in combining DTIC chemotherapy with localized high-dose (800 rads)-per-fraction radiotherapy in the treatment of unresectable metastatic melanoma. The present work investigates the radiosensitizing and chemotherapeutic properties of DTIC in an in vitro system using Chinese hamster ovary or HeLa cells and in vivo, using the KHT transplantable murine tumor. No evidence of a radiosensitizing effect of DTIC was found towards hypoxic or aerobic cells either in vitro in vivo. In vitro, high drug concentrations (1 mg/ml) were approximately 5 times more effective in killing hypoxic Chinese hamster ovary or HeLa cells than in killing aerobic cells over exposure times of 0 to 12 hr. The degree of toxicity was drug dose and temperature dependent but was not highly dependent on cell number or cell type. In vivo plasma levels of DTIC were measured with high-pressure liquid chromatography after i.p. injection of drug into C3H mice. At the highest drug doses tested, near the 50% lethal dose in mice for DTIC (0.5 mg/g), the drug was toxic to both aerobic and hypoxic tumor cells with some evidence of increased toxicity towards hypoxic cells. The present work suggests that DTIC may be more efficiently activated under hypoxic conditions as compared to aerobic conditions. The increased toxicity of DTIC under hypoxic versus aerobic conditions may prove to be a feature of this drug that can be exploited in its clinical use and in the design of new analogs of DTIC.

Animals↗
PubMed1980

Sulphydryls, ascorbate and oxygen as modifiers of the toxicity and metabolism of misonidazole in vitro.

Equimolar concentrations of cysteamine and reduced glutathione protected against the cytotoxicity of 5 mM misonidazole (MISO), whereas 5mM ascorbate enhanced its toxicity to hypoxic CHO and HeLa cells in vitro. Protection (reappearance of a shoulder region) could also be seen when cysteamine was added at later incubation times. These changes in toxicity were accompanied by changes in drug metabolism, as evidenced by radiochromatograms of cell extracts obtained after treatment with 14C-labelled MISO. In contrast, radiochromatograms obtained from cells treated with toxic levels of MISO (75 mM) under aerobic conditions indicated no drug metabolism. Both toxicity and drug metabolism could be immediately halted by introducing O2 during hypoxic exposure to MISO. These observations are discussed in terms of a possible model for the metabolism-mediated toxicity of MISO and the roles which sulphydryls and O2 may play.

Animals↗
PubMed1980

An analysis of changes in oxygen tension in stirred cellular suspensions under conditions of radiolytic and cellular consumption.

The determination and maintenance of constant known low levels of oxygen is critical in the study of chemical radiosensitization of mammalian cells in vitro. Both radiosensitization and toxicity effects must be observed, however, in systems were significant consumption of oxygen both by radiation and by cellular metabolism takes place during the experiment. We have analyzed the form of these changes in oxygen tension in stirred cellular suspensions with overlying gas phase under conditions of consumption and found it to conform to the relationship (C[t] - C infinity) = (C[o] - C infinity)e-k1t with C infinity = Cg - R/k1. Here C[t] is the oxygen tension throughout the solution, Cg is the equivalent level in the overlying gas phase, R (concentration units per second) is the depletion rate, and k1 (second-1) is a physical constant independent of oxygen concentration and depletion rate. This relationship has been confirmed in detail using a Clark-type oxygen sensor and a high-stability amplifier design due to Koch with which oxygen levels down to a few hundred parts per million can be determined with accuracy. From these data consumption rates of 4.3 +/- 0.5 ppm O2/rad by radiation in alpha-medium at 27 degrees C, and of (2.9 +/- 1.3) x 10(-17) moles O2/cell/second by metabolizing CHO cells at 37 degrees C have been determined, in agreement with literature values.

Animals↗
PubMed1979

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.

Amino Acyl-tRNA Synthetases↗