Changes in the clonogenic ability of solid tumor cells during combined exposure to hyperthermia and irradiation

A study was made of the clonogenic capacity of cells from central and peripheral zones of NKLy solid tumors of mice after heating up to 42 degrees C and after the combined effect of hyperthermia and local irradiation of the tumor. Hyperthermia markedly increased the rate of radiation death of cell populations from central tumor zones, having low oxygen tension, and had no effect on radiosensitivity of cells from peripheral well oxygenated zones. In heated tumors, the repair of potentially lethal radiation damages to cells from the peripheral zones was inhibited while in conventional irradiation conditions these damages could be restored.     

Radiosensitivity of the murine NKLy solid tumor and recovery of clonogenic cells from potentially lethal radiation damage

It was shown by the cloning method that radiosensitivity of cells of central zones was lower than that of peripheral zones (D0=6.70 and 2.91 Gy, respectively). Under conditions of recovery from potentially lethal damages radiosensitivity of clonogenic cells of peripheral zones decreased (D0=6.22 Gy) whereas that of cells from central zones changed insignificantly (D0=7.98 Gy).     

An electron spin resonance investigation of the iron-catalyzed reaction of metronidazole with cysteine

Metronidazole is used in the treatment of protozoal and bacterial infections, and has been used as a radiation sensitizer in experimental research and clinical trials. This drug is rapidly decomposed by cysteine in the presence of ferric or ferrous iron. Electron spin resonance spectroscopy demonstrates the formation of two complexes composed of iron, cysteine, and NO. The nitric oxide is probably formed by the reduction of inorganic nitrite formed by the cleavage of the metronidazole nitro group from the imidazole ring. No such reaction occurred with the 2-nitroimidazole drug, misonidazole.     

Effect of metronidazole and local irradiation of the tumor on paramagnetic metal complexes of the liver and tumor tissues

A study was made of changes in the intensity of the ESR signals in tissues of sarcoma-37 and liver of mice after radiation of the tumor, administration of metronidazole, and after the combined effect of the two factors. The most pronounced changes in the ESR signals were induced by metronidazole. An appreciable increase in the content of nitrosyl complexes in the tumor was noted after the combined effect of metronidazole and radiation which was indicative of the radiation-induced formation of a large number of metronidazole anion-radicals in the tumor.     

Chromosomal Breakage in the B. fragilis Group Induced by Metronidazole Treatment

Metronidazole is used in clinical practice for the treatment of protozoan and anaerobic infections. Under anaerobic conditions, 5-nitroimidazoles are reduced to cytotoxic nitroradicals which have been shown to act by non-specific binding to, and inactivation of the organism's DNA and enzymes. Among anaerobes, the Bacteroides fragilis group is the most relevant both in terms of frequency of isolation and antimicrobial resistance. In the present study we investigated the mechanism of action of metronidazole in the B. fragilis group. We evaluated chromosomal DNA integrity in susceptible and resistant strains during a period of 5 h after metronidazole exposure and we quantified the drug remaining in the medium after microbial growth. Metronidazole was not reduced in resistant cells which remained metabolically active and with their entire genetic material throughout the experiment. On the other hand, susceptible cells presented chromosomal breakage, a rapid consumption of dissolved metronidazole and a mortality of 90% of the bacterial population during the first 30 minutes of exposure. This interaction between metronidazole and DNA molecules that suggests strands breakage has been previously demonstrated in cell-free extract and in Escherichia coli cells. Our results show this phenomenon also occurring inBacteroides group what was not previously observed in the literature.     

The interaction of metronidazole with electron carriers of the Azotobacter vinelandii respiratory particle fraction

Abstract Metronidazole, menadione bisulfite, and oxygen oxidized NADH- and dithionite-reduced flavin of the Azotobacter vinelandii respiratory particle fraction. The oxidation of NADH-reduced flavin by metronidazole was slow compared to the oxidations by menadione bisulfite and oxygen. Metronidazole caused spectral changes characteristic of cytochrome d oxidation, but the changes in NADH-reduced particles were slow.     

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.     

Radiation response of clonogenic cells of solid tumors. Clonogenic capacity of the murine NKLy solid tumor and its oxygen status

Clonogenic capacity and oxygen tension were determined in peripheral and central zones of solid tumors NKLy in vivo. With low pO2 in central zones (2.69 +/- 0.60 as opposed to 4.64 +/- 0.68 mm m. c. in peripheral zones) and in the presence of anoxic sites, the effectiveness of cloning, with inoculation immediately after isolation of cells from central zones, did not differ from that from peripheral ones (6.36 +/- 0.45 and 6.78 +/- 0.68%, respectively).     

Effect of metronidazole on the course of experimental anaerobic streptococcal pneumonia

The effect of metronidazole in experimental anaerobic pneumonia in guinea pigs infected with peptostreptococci has been studied. In the untreated animals the prolonged pathological process in the lungs is mainly associated with the suppression of the functional state of the thymus-dependent link of the immune system. Metronidazole suppresses anaerobic flora, stimulates local immune reaction and arrests the development of inflammatory changes in the lungs. The drug eliminates the unbalance in some subpopulations of immunocompetent cells and restores the number of cells regulating immune response to the normal level. The results obtained in this investigation make it possible to recommend the trial of metronidazole in the complex therapy of peptostreptococcal pneumonia.     

The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis

The nitro radical-anion of the anti-trichomonal drug metronidazole has been detected by electron spin resonance spectrometry under anaerobic conditions in suspensions of intact hydrogenosomes isolated from the parasitic protozoon Trichomonas vaginalis. Metronidazole reduction was driven by pyruvate, but progressive damage to the radical generating system was observed. Quenching of signals due to metronidazole radicals by chromium oxalate suggests that the radicals generated within the organelle can cross the hydrogenosomal membrane into the external medium. Even if a similar process of radical migration occurs in vivo, it seems likely that intrahydrogenosomal damage may explain drug action.     

Metronidazole in prevention and treatment of bacteroides infections after appendicectomy.

The frequency of non-clostridial anaerobic infection was studied in 95 patients who had undergone acute appendicectomy: 49 received prophylactic metronidazole and 46 received placebo. Anaerobic infection did not develop in any of the metronidazole-treated patients, but infections did develop in nine (19%) of the 46 controls. Metronidazole is conveniently administered by suppository to patients who cannot take oral drugs. Five patients with intra-abdominal infections caused by non-clostridial anaerobes were successfully treated with metronidazole.     

Molecular weight and quaternary structure of ribulose bisphosphate carboxylase from the cyanobacterium,Synechococcus sp.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH ₄ ⁺ . Light-dependent C₂H₂ reduction and H₂ production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H₂-palladised asbestos prior to assay, it only partially restricted C₂H₂ reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H₂-dependent photoreduction of ¹⁴CO₂; (c) -glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor).Metronidazole (1 mM) completely inhibited C₂H₂ reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C₂H₂ reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.     

On the mutagenicity of nitroimidazoles

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.     

Paraendocervical route for metronidazole administration in complex treatment of chronic Trichomonas endocervicitis]

The data on paraendocervical administration of metronidazole in the treatment of patients with relapsing Trichomonas endocervicitis are presented. Metronidazole was administered as 0.5% solution in a dose of 0.04 g once a day for 8-10 days in complex traditional therapy including oral use of metronidazole and immunocorrigating and local treatment. It was shown that paraendocervical administration of the protistocidal agent provided earlier regression of the urogenital symptoms and 2.8 times lower frequency of the relapses.     

Connective matrix organization in chronic granulomas of experimental paracoccidioidomycosis

The histological and ultrastructural aspects of chronic granulomas from rats infected intraperitoneally with Paracoccidioides brasiliensis are described with special emphasis on the composition of the extracellular matrix.The granulomas were structurally arranged in two zones, one central containing fungi, and the other peripheral.The extracellular matrix was composed of collagen types I and III, proteoglycans, glycoprotein, and an undefined amorphous substance. The main cellular population was represented by macrophages, epithelioid cells, and giant cells in the central zone, and fibroblasts in the peripheral zone.The fibrotic process was a critical event in this stage of the infection, and showed a centrifugal direction. This might be provoked by direct stimulus from the fungi or by macrophage-fibroblastic interaction.     

Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase

Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.     

Nitroimidazole action in Entamoeba histolytica: a central role for thioredoxin reductase

Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.     

Alaska sentinel surveillance for antimicrobial resistance in Helicobacter pylori isolates from Alaska native persons, 1999-2003

BACKGROUND: Previous studies in Alaska have demonstrated elevated proportions of antimicrobial resistance among Helicobacter pylori isolates. MATERIALS AND METHODS: We analyzed H. pylori data from the Centers for Disease Control and Prevention (CDC)'s sentinel surveillance in Alaska from July 1999 to June 2003 to determine the proportion of culture-positive biopsies from Alaska Native persons undergoing routine upper-endoscopy, and the susceptibility of H. pylori isolates to metronidazole [minimum inhibitory concentration (MIC) of > 8 g metronidazole/mL), clarithromycin (MIC > or = 1), tetracycline (MIC > or = 2) and amoxicillin (MIC > or = 1)] using agar dilution. RESULTS: Nine-hundred sixty-four biopsy specimens were obtained from 687 participants; 352 (51%) patients tested culture positive. Mean age of both culture-positive and culture-negative patients was 51 years. Metronidazole resistance was demonstrated in isolates from 155 (44%) persons, clarithromycin resistance from 108 (31%) persons, amoxicillin resistance from 8 (2%) persons, and 0 for tetracycline resistance. Metronidazole and clarithromycin resistance varied by geographic region. Female patients were more likely than male subjects to show metronidazole resistance (p < .01) and clarithromycin resistance (p = .05). CONCLUSIONS: Resistance to metronidazole and clarithromycin is more common among H. pylori isolates from Alaska Native persons when compared with those from elsewhere in the USA.     

Trichomoniasis in a Closed Community: Efficacy of Metronidazole

A retrospective survey of women treated in prison for trichomonal vaginitis with metronidazole showed that 488 of 496 (98·3%) were cured after one course of drugs. Five of the eight treatment failures were successfully treated by further courses of metronidazole.A regimen of 400 mg metronidazole twice daily for seven days is simple and effective when taken in prescribed dosage. Metronidazole is still the drug of choice for trichomonal vaginitis. No toxic reactions were observed and there was no evidence that the drug has lost efficacy in the last ten years.     

Pathologic anatomy and CT correlations in the glioblastoma multiforme

6 cases of glioblastoma multiforme, 1 anaplastic astrocytoma, and 1 astrocytoma were studied pathologically post mortem. The specimens were compared to antemortem CT scans, when available. By use of whole brain sections, each glioblastoma was divided into a central area of necrosis, a surrounding rim of hypercellular neoplasm, and a peripheral zone of infiltration. The dimensions and areas of these three zones were quantified. The striking findings of the study were the often small diameter of the hypercellular region and the frequently narrow peripheral zone of detectable infiltrating cells. Comparison of the autopsy specimens with CT scans confirmed previous studies of the glioblastoma. The radiographic central area of low density is necrosis; the enhancing rim is a markedly hypercellular region, and the perilesional low density encompasses a population of infiltrating tumor cells.