Concentrations Of Bleomycin Research Articles

Potentiation of bleomycin cytotoxicity in Saccharomyces cerevisiae.

Lesions introduced into cellular DNAs prelabeled with [2-14C]thymidine or [6-3H]thymidine, as well as cell killing, were inhibited by the presence of EDTA during 20-min reactions of Saccharomyces cerevisiae cells with the low-molecular-weight bleomycin family of anticancer antibiotics. In contrast, the level of killing by low concentrations of bleomycin was higher among cells which had grown for three generations in defined synthetic complete medium supplemented with ferrous sulfate than among cells grown without iron supplementation. In S. cerevisiae, the uptake of iron is facilitated by a plasma membrane ferric reductase activity and a high-affinity (Km = 5 x 10(-6) M) ferrous uptake system. Lethal effects of 1.3 x 10(-6) M bleomycin increased approximately 50% with 10(-5) M Fe(II), nearly twofold with 10(-4) M Fe(II), and 2.8 times with 10(-3) M Fe(II). Thus, iron preloading is a new experimental approach to increasing and studying the effects of the glycopeptides on cellular DNAs and other cellular targets. This approach could also be used for studying and better understanding DNA repair genes and could serve as a model for studies of redox active chemicals in biological systems.

Potentiation of bleomycin by the aminothiol WR-1065 in assays for chromosomal damage in G 0 human lymphocytes

The aminothiol radioprotector WR-1065 potentiates the induction of chromosome aberrations and micronuclei by the chemotherapy drug bleomycin in G 0 human lymphocytes. Potentiation by 5 mM WR-1065 was observed at bleomycin concentrations from 0.1 to 100 μg/ml in a 2-h treatment. The frequencies of micronuclei induced by bleomycin in the presence of WR-1065 reached that of 500-fold higher concentrations of bleomycin alone. The potential therapeutic implications of these findings are discussed.

NAD depletion after in vitro exposure of murine lung slices to bleomycin

Bleomycin (BLM), a DNA-cleaving, antitumor antibiotic, causes pulmonary fibrosis. It also causes cell injury and activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP; EC 2.4.2.30) in lung slices exposed to the drug in vitro. 3-Aminobenzamide (3-AB), aPAP inhibitor, prevents enzyme activation and cell injury. We have examined the potential role of ATP and NAD depletion in injury of BLM-sensitive C57B1/6N and -resistant BALB/cN murine lung slices treated with BLM or deprived of glucose, the major metabolic substrate of lung. Lung slices either were treated for 45 min with injurious concentrations of BLM (10–500 μg/mL) or were incubated without glucose, in the presence or absence of 2.5 mM 3-AB. Only the highest concentration of BLM, 500 μg/mL, caused any ATP depletion, and this 35% decrease was transient, occurring at 220 min in C57B1/6N slices. In contrast, glucose deprivation caused 50–70% ATP depletion in slices from both strains. BLM alone at 100 and 500 μg/mL caused a sustained 30–70% NAD depletion from 75 min through 400 min in C57B1/6N mouse lung slices. In the resistant BALB/cN lung slices, NAD depletion by BLM was only seen at 400 min. 3-AB almost completely antagonized NAD depletion in slices from both strains. In contrast to BLM, glucose deprivation did not decrease NAD levels unless 3-AB was present in C57B1/6N slices. Thus, ATP depletion may play a role in the injurious effects of glucose deprivation, but does not appear to be a major factor in pneumocyte injury caused by BLM. NAD depletion or other effects of PAP activation appear to account for the strain-selective, injurious effect of BLM on lung tissue.

Effects of Bleomycin with Iododeoxyuridine, 5′-Amino-5′-Deoxythymidine, and Radiation in a Human Bladder Cancer Cell Line

In vitro bleomycin cytotoxicity in 647V, a human bladder cancer cell line, is enhanced when the cells are preincubated for one cell cycle (24 h) with clinically relevant concentrations (2 microM) of iododeoxyuridine (IdUrd). Bleomycin cytotoxicity after a 1-h exposure is further enhanced by a 24-h preincubation with 2 microM IdUrd plus 30 microM 5'-amino-5'-deoxythymidine (5'-AdThd). Chemosensitization of clinically achievable plasma levels of bleomycin (1-10 mU/ml for 1 h) by IdUrd (+/- 5'-AdThd) is associated with the level of DNA incorporation of IdUrd in 647V cells and with enhanced bleomycin-induced damage in IdUrd-substituted DNA compared to control DNA, as measured by rapid alkaline elution. We also studied the in vitro effects of bleomycin (+/- IdUrd) treatment on the subsequent radiation response of 647V cells. We found that a 1-h preirradiation exposure of 647V cells to low concentrations of bleomycin (1 mU/ml) can radiosensitize the cells with an even greater interaction exhibited by pretreatment with 2 microM IdUrd (24 h) plus bleomycin (1 h) vs control. Since the principal normal tissue toxicities of IdUrd (involving bone marrow and gut) and bleomycin (involving lung) do not overlap, these in vitro data suggest that chemosensitization of bleomycin by IdUrd may provide a new therapeutic regimen for chemotherapy and a significant enhancement for radiotherapy in human bladder cancer and possibly other epithelial cancers.

Histologic, pharmacologic, and immunocytochemical effects of injection of bleomycin into viral warts

The plasma concentration of bleomycin after injection of bleomycin into warts is unknown, as is the long-term stability of bleomycin solution. Our purpose was to measure plasma bleomycin concentration after injection of bleomycin into warts, to relate histologic and immunocytochemical changes in warts to possible mechanisms of action of bleomycin, and to asses the long-term stability of stored frozen bleomycin solution. One milligram of bleomycin was injected into warts on the hands of seven men. Blood samples were taken 15 to 120 minutes after injection, and plasma bleomycin was measured by radioimmunoassay. Warts were removed 2 hours and 48 hours after treatment and studied histologically by light microscopy and for the presence of bleomycin by immunocytochemistry. The bleomycin concentration in 8 aliquots of solution stored at -20 degrees C for varying periods was measured by radioimmunoassay. Peak levels of bleomycin of 7 to 113 ng/ml were reached by 45 minutes after injection. Plasma bleomycin exposure ranged from 515 to 5137 ng/ml/min between 15 and 120 minutes after injection. The most pronounced histologic changes at 48 hours were individual keratinocyte apoptosis throughout the epidermis merging into areas of complete epidermal necrosis, diffuse neutrophil accumulation, and microabscess formation at the granular layer. Immunocytochemistry demonstrated tissue-fixed bleomycin in all levels of the epidermis except the basal layer and most prominently in the granular layer. Bleomycin in solution stored for up to 27 months at -20 degrees C in glass showed no significant loss of immunoreactivity. The use of bleomycin for the treatment of warts results in significant systemic drug exposure; thus it would be prudent to exclude pregnancy before treating women of child-bearing age. Bleomycin probably has a direct toxic effect on keratinocytes. Dilute bleomycin solution stored at -20 degrees C in glass is stable.

Chromosome damage induced by nanomolar concentrations of bleomycin in porated mammalian cells

We have examined chromosome damage caused by a wide range of bleomycin (BLM) concentrations in Chinese hamster ovary (CHO-K1) cells reversibly porated by the bacterial cytotoxin streptolysin-O (SLO). Chromosome damage was measured using the micronucleus cytokinesis block technique (employing cytochalasin-B). Treatment of exponentially growing cells with 0.045 IU/mL SLO for 5 min resulted in up to a thousand-fold and a million-fold increase in biological effectiveness, compared to treatment in the absence of SLO for 24 hr and 5 min, respectively. Increases in micronuclei of 4–5 times background level were observed after only 5 min exposure to the drug in the presence of SLO at doses as low as 100 pg/mL (≈70 pmol/L). These results indicate that the use of SLO may facilitate the treatment of cells with BLM for periods of time resembling acute exposure to ionizing radiations.

Heat sensitivity of bleomycin-sensitive CHO derivatives is not due to improper initialization of heat shock response.

We have investigated whether differences in the heat shock response exist between CHO and three bleomycin-sensitive, heat-sensitive CHO derivatives. The binding of heat shock factor (HSF) in response to heat and varying concentrations of bleomycin in the four cell lines was examined using a gel shift assay and a synthetic heat shock element (HSE). Heat (45 degrees C, 10 min) and exposure to 1 micrograms/ml bleomycin for 1 h at 37 degrees C induced similar levels of HSF binding in all four cell lines. We also examined if bleomycin dose and the length of recovery from bleomycin treatment affected the induction of HSF binding. The level of activated HSF binding to HSE was higher in cells treated with low doses (1 ng/ml) of bleomycin than in cells treated with 1 or 25 micrograms/ml bleomycin. The amount of activated HSF was directly proportional to the time elapsed since bleomycin treatment. Our results therefore indicate no difference between CHO and its bleomycin-sensitive derivatives in the ability to initiate the heat shock response as determined by the production of activated HSF in response to either heat or bleomycin. We conclude that the intrinsic thermosensitivity of these cell lines is not related to the early response to heat shock, but either occurs later in the pathway or is unrelated to events after heating.

Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways.

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes. Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations. RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.

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This study was undertaken to examine whether the production of active oxygen species by bleomycin (BLM) could be enhanced by various cytokines in human neutrophils.Peripheral blood neutrophils were obtained from normal volunteers, and the production of superoxide radical (O-2) and the generation of luminol dependent chemiluminescence (LDCL) were measured by the cytochrome C reduction method and an ATP photometer, respectively. IL-1α, IL-1β, IL-2, IL-6, IL-8, G-CSF, IFN-γ, and TNF-α were used as cytokincs, and neutrophils were treated with these cytokines for 10 minutes at 37°C. Phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as stimulants of O-2 and LDCL production, respectively.The results of these experiments revealed slight production of O-2 from neutrophils by BLM, and the production of O-2 by PMA was enhanced by BLM concentrations over 0.3 μg/ml. With a concentration of 2, 500 U/ml of IFN-γ, and at concentrations over l ng/ml of TNF-α, small amounts of O-2 wcre produced without the stimulants or BLM. Thc production of O-2 by BLM was enhanced by IL-1α, IL-1β, IL-8, G-CSF, IFN-γ and TNF-α, that produced by PMA was enhanced by IFN-γ or TNF-α, and O-2 produccd by BLM and PMA in combination was enhanced by IL-8, G-CSF, IFN-γ and TNF-α.These results suggest that the production of active oxygen species by BLM in vivo may be enhanced by these cytokines, except for IL-2 and IL-6. Through this mechanism, cytokines might enhance the antitumor activity of BLM.

Cytomegalovirus-enhanced induction of chromosome aberrations in human peripheral blood lymphocytes treated with potent genotoxic agents.

Human cytomegalovirus (HCMV) has been shown to increase the frequency of chromosome aberrations, primarily chromatid-type, in human peripheral blood lymphocytes (PBLs). Because HCMV persists in most humans, pathologically activates cells, and may perturb the cell cycle, we investigated the possibility that HCMV-infected cells have a modified sensitivity to chromosome damage induced by genotoxic chemicals. Uninfected PBLs exposed to bleomycin (3 to 100 micrograms/ml) demonstrated a linear increase in the frequency of chromosome aberrations. HCMV infection of PBLs at an intensity that did not cause detectable damage followed by exposure to the same concentrations of bleomycin resulted in a significant enhancement (p less than 0.01) in the frequency of chromosome aberrations relative to the effect of bleomycin alone. A more than additive enhancement of the frequency of chromosome aberrations was also noted in HCMV-infected PBLs exposed to 4-hydroxyaminoquinoline-1-oxide (4-HAQO; 0.1 to 0.3 micrograms/ml) relative to uninfected cells treated with 4-HAQO alone. No increase in the percentage of aberrant cells or the frequency of chromosome aberrations was observed in HCMV-infected cells treated with 4-nitroquinoline-1-oxide (4-NQO) relative to similarly treated uninfected PBLs. These results suggest that HCMV can potentiate the induction of chromosome aberrations in human PBLs caused by potent DNA damaging agents.

In vivo measurements of the fraction of dose of bleomycin labeled with cobalt 57 delivered to human tumors

Concentrations of bleomycin labeled with cobalt 57 (Co-bleo) over time were measured in vivo in 17 patients with 32 sites of lymphoma and 18 patients with lung tumors after administration of the same dose of bleomycin. There were marked variations in individual tumor drug concentrations even among tumors with the same histologic type, indicating that the tumor concentration of this drug in individuals cannot be predicted from the administered dose. Also, tumor concentration could not be predicted from the area under the concentration over time curve (AUC) of Co-bleo in the blood; there was no correlation (r = 0.53) between the AUC and the concentration in the tumor at any point in time between 30 minutes and 8 hours after injection. There was no significant difference in the percent of the injected dose per milliliter (%ID/ml) which was delivered to the tumor when low and high amounts of bleomycin were administered to the same patient. Also, a good correlation (r = 0.88) between the %ID/ml over time was found when injection of low and high doses of bleomycin were compared. The results indicate that using quantitative single photon emission computed tomography (SPECT) and a labeled tracer dose it is possible to predict what fraction of the dose of a chemotherapeutic drug will concentrate in an individual patient's tumor in vivo. They also show that, for bleomycin, escalation of dose will result in a proportional increase of tumor concentration. This increase depends on individual properties of tumors which can be measured quantitatively in vivo by SPECT and are expressed as percent of %ID/ml of tumor tissue.

Inhibition of bleomycin-induced cellular DNA strand scission by 1,10-phenanthroline

Inhibition by 1,10-phenanthroline of cellular DNA strand scission induced by the antitumor antibiotic bleomycin in Ehrlich ascites tumor cells was studied. DNA alkaline elution was performed on cells after 1-hr bleomycin treatments. Pretreatment for 24 hr with initial 1,10-phenanthroline concentrations of 0.2 nmol/10 5 cells, which depletes cells of ferritin iron by 80%, had no consistent effect on bleomycin strand breakage. However, simultaneous treatment with 3.1 nmol of 1,10-phenanthroline/10 5 cells and with bleomycin concentrations from 5 to 25 μM decreased both apparent double-stranded breaks and random breakage. When cells were treated with both 3.1 nmol of 1,10-phenanthroline/10 5 cells and 25 μM bleomycin, washed free of both drugs, and incubated at 37° for 1 hr, the resulting breakage was equivalent to that found in cells treated with bleomycin only. When the combination treatment was extended to 4 hr, cell washing and reincubation resulted in increased strand scission, as compared with strand scission in cells treated with bleomycin only. Growth inhibition by bleomycin was not affected appreciably by temporary suppression of DNA strand breakage activity.

Analysis of bleomycin‐induced mutagenic functions related to the PSO4 (= XS9) gene of saccharomyces cerevisiae

In the present work we analyse some properties of the pso4-1 (= xs9) mutant of the yeast Saccharomyces cerevisiae, which is blocked in both reverse mutation and recombination. We have applied the "apparent survival test" as a method to characterize putative inducible error-prone repair activities related to the PSO4 gene. As the mutagenic agent we have used bleomycin, which is an antitumour radiomimetic antibiotic. Survival, reverse mutation yield, and reverse mutation frequency were determined as a function of bleomycin concentrations (1-60 micrograms/ml) in the logarithmic phase of growth. It is shown that the PSO4 gene product is poorly involved in bleomycin sensitivity and that reverse mutation induced by this mutagen is dramatically reduced in the pso4-1 lys2 mutant strain, as compared with the isogenic SC7K (lys2) strain. M(x) functions exhibit linear-quadratic courses for both the SC7K (lys2) and the pso4-1 lys2 strains. The apparent survival functions display "humps" in both strains, corresponding to the resistant components of the survival functions. These facts suggest that the mutagenicity of bleomycin depends on at least one inducible error-prone repair pathway and that the PSO4 gene product could act as a mutation triggering factor.

The kinetics of DNA damage by bleomycin in mammalian cells

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1–5 μg/ml) and short treatments (5–15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.

Comparative flow cytometric analysis of bleomycin toxicity on normal and tumour sheep cell kinetics in vitro

The cytotoxic effects of bleomycin on cell proliferation, morphology and cycle progression were compared in a sheep tumour cell line and in normal sheep sinus cells. They were concentration and time dependent, irreversible, and were accompanied by increased cell diameter and cytosolic synthetic activity. Cell accumulation in the G2M phase, without much change in the cell cycle progression through the S phase, was observed using flow cytometry. Although the tumour cells showed a greater sensitivity to low bleomycin concentrations compared to the normal cells, both cell types exhibited equal sensitivity to higher drug concentrations. Provided that tumour cells are also preferentially affected by bleomycin in vivo, these findings raise the possibility that bleomycin cytotoxicity can be reduced or avoided in normal tissue.

Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin

Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01–0.1 μg/ml, for 48 h and then treated with a high concentration (1.5 μg/ml) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromocomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.

Bleomycin hypersensitivity in dyskeratosis congenita fibroblasts, lymphocytes, and transformed lymphoblasts

We studied the responses of several dyskeratosis congenita (DC) cell lines to the DNA strand-cleaving and base-damaging agent bleomycin. Fibroblasts, peripheral blood lymphocytes, and transformed lymphoblasts of six DC patients and an obligate DC heterozygote showed more chromatid breaks than did respective controls exposed to various concentrations of bleomycin during the G2 phase of the cell cycle (P less than 0.0001). Unsynchronized DC fibroblasts in culture also showed decreased survival, compared to normals, following bleomycin treatment. DC lymphocytes treated with bleomycin for the final 24 h of culture showed more chromatid- and chromosome-type damage than did normals (P less than 0.0001) or G0-treated DC lymphocytes. Spontaneous chromosome breakage was normal in all six DC cell lines. The ability to distinguish affected and heterozygous DC cells without spontaneous chromosome instability from normals on the basis of their bleomycin hypersensitivity provides a marker for future studies of the pathogenesis of this disorder.

Effect of 3-aminobenzamide on chromosome damage in human blood lymphocytes adapted to bleomycin

Human blood lymphocytes, pre-treated with very low (adaptation) concentrations of bleomycin for 48 h and then exposed to a high (challenge) dose of the same agent or X-rays, became significantly less sensitive to the induction of chromosome damage than those which did not receive the pre-treatment, indicating an induction of 'adaptive repair' process. This repair process was negated when 3-amino-benzamide, an inhibitor of poly (ADP) polymerase, was added to the cultures immediately after the challenge treatment. The magnitude of negation in the adaptation response was greater in the case of lymphocytes challenged with X-rays as compared with those challenged with bleomycin.

Application of radioimmunoassay to monitor treatment of human cerebral gliomas with bleomycin entrapped within liposomes.

A radioimmunoassay was assessed for its suitability for monitoring the blood and urinary concentrations of bleomycin entrapped within large unilamellar liposomes in patients receiving therapeutic doses of the drug. Bleomycin entrapped within liposomes was administered by direct intracerebral injection to three patients with grade III to IV cerebral gliomas, twice weekly for up to six weeks. No clinically important toxic effects were observed which could have been attributed to these preparations, although all three patients showed progressive deterioration in their clinical condition. This type of treatment may be more beneficial to patients at an earlier stage of their disease.

The pharmacology of intraperitoneally administered bleomycin.

The clinical pharmacology of intraperitoneally administered bleomycin was examined in 11 patients with malignancies confined predominantly to the peritoneal cavity. Bleomycin (60 mg/m2) was mixed in 2 L of 0.9% NaCl, and administered rapidly into the peritoneal cavity via a Port-a-Cath (Pharmacia Nu Tech, Inc, Walpole, MA). The cavity was drained following either a four- or eight-hour dwell time. Bleomycin concentration in the peritoneal cavity and plasma was determined by radioimmunoassay. A total of 15 courses was studied. The peak peritoneal: plasma concentration ratio averaged 22, and total exposure for the peritoneal cavity averaged seven-fold greater than that for the plasma. The peritoneal and plasma half-lives for bleomycin were 3.2 and 6.0 hours, respectively. The peritoneal clearance of bleomycin averaged 11.3 mL/min/m2. Abdominal pain occurred on 47% of courses; other toxicities included fever, skin rash, and mucositis. We conclude that there is a substantial pharmacologic advantage to the peritoneal route for tumors confined to the peritoneum, but that the advantage is less, and the local toxicity greater, than that found with the intraperitoneal administration of many other anticancer drugs.