Medically inoperable peripheral lung cancer treated with stereotactic body radiation therapy.

BACKGROUND: Lung cancer is the most frequent cause of cancer-related death in North America. There is wide variation between patients who are medically inoperable and those managed surgically. The use of stereotactic body radiotherapy (SBRT) has narrowed the gap in survival rates between operative and non-operative management for those with early stage disease. This retrospective study reports outcomes for the treatment of peripheral non-small cell lung carcinoma (NSCLC) with SBRT from a single community practice. METHODS: Sixty-seven consecutive patients (pts) with inoperable, untreated peripheral lung tumors were treated from 2010 through 2012 and included in this study. Stereotactic targeting was facilitated by either spine or lung-based image guidance, either with or without fiducial marker tracking with a frameless robotic radiosurgery system. Peripheral tumors received a median biological effective dose (BED) of 105.6 Gy10 or in terms of a median physical dose, 48 Gy delivered over 4 daily fractions. Survival was measured using the Kaplan-Meier method to determine rates of local control, progression of disease and overall survival. The Cox proportional hazards regression model was used to study the effects of tumor size, stage, histology, patient age, tumor location (lobe), tracking method, and BED on the survival distributions. RESULTS: The median follow-up for this cohort was 24.5 months (range: 2.4-50.3) with an overall (OS) 3-year survival of 62.4 % (95 % CI: 74.3-47.3). The median progression-free survival was 28.5 months (95 % CI: 15.8 months to not reached). Local control (LC), defined as a lack of FDG uptake on PET/CT or the absence of tumor growth was achieved in 60 patients (90.9 %) at the time of first follow-up (median 3 months, range: 1-6). Local control at one year for the entire cohort was 81.8 % (95 % CI, 67.3-90.3). The one-year OS probability among those who achieved local control at first follow-up was 86.2 % (95 % CI, 74.3-92.9) but no patients who did not achieve LC at first follow-up survived one year. Of the 60 pts that achieved initial LC, 16 have died. The rates of local control, progression-free survival and overall survival were not statistically different for patients treated using a fiducial target tracking system versus non-invasive guidance. (p = 0.44, p = 0.97 and p = 0.66, respectively). No National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE-4) grade 3 or greater toxicity was observed. CONCLUSION: SBRT is an effective treatment for medically inoperable NSCLC patients with peripherally located tumors. This therapy appears to be well tolerated with low toxicity, and patient outcomes when using non-invasive tumor tracking systems are not inferior to traditional fiducial-based techniques.

Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens.

Coccidiosis of chickens is an economically important disease caused by infection with species of Eimeria. The oocysts of some of the seven recognized species are difficult to distinguish morphologically and for this reason diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria. The real-time PCR provides both sensitivity and speed for the analysis of DNA samples, and the approach has the capability of quantifying DNA. Together with a protocol for the extraction of DNA directly from faecal samples, real-time PCR assays have been established for the detection and quantification of seven species of Eimeria that infect chickens in Australia. The assays target one genetic marker, the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2), use TaqMan MGB technology with species-specific probes, and can be multiplexed in pairs such that the seven species of Eimeria can be screened in four reaction tubes. A test screen of commercial flocks identified more Eimeria-infected chickens than were detected by coproscopic examination for oocysts. These molecular assays can also be used for the quality control of mixed-species vaccines. The ability to multiplex the assays makes them particularly practical for screening samples from chickens with mixed-species infections where the relative abundance of each Eimeria species present is required.

Association of redox-active iron bound to high molecular weight structures in nuclei with inhibition of cell growth by H2O2.

Perturbations to Fe species contributing to generation of DNA single-strand breaks (SSBs) and inhibition of growth by H2O2 were studied in HL-60 cells made Fe-deficient by 24 h pretreatment with 144 microM bathophenanthroline disulfonic acid and 400 microM ascorbic acid (Free Radic. Biol. Med. 20: 399; 1996). The diffusion distance for SSB generation (d) in Fe-deficient cells, measured via inhibition with the *OH scavenger Me2SO using alkaline elution, was 6.5 nm. This is similar to the d for Fe-normal cells reported previously. After 1 and 3 h in fresh RPMI 1640 medium containing 10% serum, SSB generation increased from 29 to 56 and 93% of control Fe-normal cells, respectively. The d of the major contributor to SSB generation at these two treatment times was 1.9 nm. This d resembled the d for Fe-ATP as determined in isolated Ehrlich cell nuclei. The association of ATP with Fe2+ was further supported by decreased SSB generation in cells in which ATP synthesis was inhibited. In contrast to SSB generation, H2O2-induced inhibition of growth of Fe-deficient cells treated immediately after placing in fresh medium was not appreciably different from Fe-normal cells. However, after 3 h, an approximately 70% greater concentration of H2O2 than for control, Fe-normal cells was required to inhibit growth. This increase in H2O2 concentration was associated with decreased generation of SSBs by H2O2 in isolated HL-60 cell nuclei. Thus, Fe bound to nuclear structures is more closely associated with inhibition of cell growth than apparent Fe-ATP species. In parallel experiments, changes in total cellular Fe assayed by ashing and complexing with ferrozine were consistent with a non-transferrin mode of acquisition. These short-term changes appear due to processes accompanying reestablishment of the Fe content and distribution normally observed during long-term growth.

Diffusion distances of known iron complexes in model systems.

Diffusion distances (abbreviated d's), the distances between the sites of generation of presumed hydroxyl radicals (*OH) by low molecular weight forms of Fe and the site of their reaction with substrate, were measured for three model systems for cellular DNA of varying degrees of complexity. Two d's for Fe complexed with each of ethylene diamminetetraaccetic acid (FeEDTA) and nitrilotriacetic acid (FeNTA) were measured for generation of malondialdehyde-type products (MDA) from deoxyribose and of single-strand breaks (SSBs) in the plasmid pBR322. The closer d's for pBR322 SSB generation (5-6 nm) were considerably greater than the d's for MDA generation in the deoxyribose assay (2-3 nm). This is consistent with charge-charge interactions playing an important role in defining d. The d's for FeNTA, FeEDTA, and other Fe species generating SSBs in isolated Ehrlich ascites tumor cell nuclei ranged from 2.1 to 14 nm. Charge-charge interactions, Fe-ligand-specific interactions, and binding to nuclear components were concluded to be important factors affecting d in isolated nuclei. Other factors related to nuclear structure may also play a role.

Protection of DNA in HL-60 cells from damage generated by hydroxyl radicals produced by reaction of H2O2 with cell iron by zinc-metallothionein.

Scavenging of hydroxyl radicals (.OH) by the zinc form of metallothionein (ZnMT) was studied in HL-60 cells and in nuclei from such cells previously treated with ZnCl2 (ZnMT cells). Cells were grown for 48 h to label DNA for alkaline elusion experiments. During the last 24 h 0.1 mM ZnMT was included to induce ZnMT. Generation of DNA single-strand breaks (SSBs) by H2O2 in cells (5 x 10(5)/ml) treated at 4 degrees was increased by approximately 70% in Zn-treated cells by comparison with control cells. These cells had grown from an initial concentration of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cells started at 6 x 10(5)/ml and growing to a final concentration of 20 x 10(5)/ml did not exhibit a similar increase in SSBs. This elevation in SSBs was traced to an increase in cell Fe content which exhibited a sharp dependence upon concentrations of cells and of ZnCl2 at the time of addition. The diffusion distance (d) from Fe to DNA of ZnMT cells treated with H2O2 was found to be 3.4 nm. This compares with a distance of 6.1 nm in control cells. SSB generation by hydroxyl radicals formed by 137Cs-gamma rays in Zn-treated cells decreased by 12%, accompanied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentially reacts with OH formed at some distance from DNA. In nuclei isolated from ZnMT cells started at 5 x 10(5)/ml, SSB generation by H2O2 increased by 60%. The d in these nuclei was 4.9 nm, similar to the distance in control nuclei reported previously. These data suggest that, in addition to altering the scavenging environment, treatment of cells with Zn leads to an increase in reactive Fe in cells and in isolated nuclei which can generate DNA damage through reaction with H2O2.

Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites.

The reaction of the sulfhydryl groups in metallothionein with hydrogen peroxide was examined in HL-60 cells. Partial purification of cell cytosol using Sephadex G-75 chromatography showed that zinc-metallothionein (Zn-MT) was induced by 24-h treatment with 100 microM ZnCl2, but the cellular glutathione content and glutathione peroxidase and catalase activities were unaffected. The ratio of H202 concentrations needed to reduce cell survival 50% in Zn-induced cells compared to normal cells was 1.65 to 1. According to alkaline elution experiments, the average ratio of single-strand breaks caused by H202 at 37 degrees C in Zn-induced vs normal cells was 0.5 to 1. A similar reduction in strand breakage was seen in nuclei from Zn-treated cells exposed to H202; however, at 4 degrees C protection against DNA strand breakage by Zn pretreatment was not seen. Incubation of Zn-pretreated cells with H202 at 37 degrees C but not 4 degrees C was accompanied by loss of Zn bound to MT and a reduction in the number of MT sulfhydryl groups. In the absence or presence of Zn-MT, sulfhydryl groups from glutathione and protein fractions were also reduced by exposure of cells to H202. However, thiolate groups in the MT fraction were preferentially lost compared to the other pools of sulfhydryl residues. Zn-MT also spared glutathione sulfhydryl groups in vitro from oxidation by H202. Protection against strand breakage correlated with the ability of Zn-MT to react in vitro with H202 at 37 degrees C, but not at 4 degrees C. The reaction was slow and was not inhibited by the presence of an hydroxyl radical scavenger, dimethyl sulfoxide. Similarly, in cells dimethyl sulfoxide did not prevent the loss of sulfhydryl groups from glutathione or protein. Incubation of MT or higher molecular weight fractions from cells exposed to H202 with either 2-mercaptoethanol or dithiothreitol in the presence of Cd failed to regenerate any detectable, reduced MT, suggesting that MT sulfhydryl groups were oxidized by H202 beyond the disulfide oxidation state.

Inhibition of hydroperoxide-induced DNA single-strand breakage by 1, 10-phenanthroline in HL-60 cells: implications for iron speciation.

The speciation of intracellular iron which reacts with hydroperoxides to cause DNA damage was investigated in HL-60 cells using 1,10-phenanthroline (OP) to compete with ligands for iron. HL-60 cells were treated with various concentrations of OP for 30 min at 37 degrees C followed by 30 min treatment with H2O2 or t-butylhydroperoxide (tert-BuOOH) at 4 degrees C. Single-strand breaks (SSBs) were measured by alkaline elution. OP (5 microM) completely inhibited production of SSBs by tert-BuOOH. This contrasts with results for H2O2, for which it was not possible to inhibit all SSBs even at OP concentrations up to 60 microM. Induction of SSBs by tert-BuOOH decreased according to a SSB:OP ratio of 1:3.28 +/- 0.23 (+/-1 SE). This is consistent with inhibition achieved by occupation of all coordination sites on iron by OP, such as by formation of Fe(OP)3. In contrast, after subtraction of the noninhibitable fraction, SSBs induced by H2O2 decreased according to a 1:1.53 +/- 0.26 ratio. This suggests that inhibition of H2O2-induced SSBs is partially or wholly achieved by formation of different structures than Fe(OP)3. Tert-BuOOH did not cause SSBs in nuclei isolated from HL-60 cells. However, H2O2 induced SSBs which were inhibited 45 +/- 8% (+/- 1 SD) by dimethyl sulfoxide. Analysis of the concentration dependence of inhibition by dimethyl sulfoxide indicated that the H2O2-reactive iron in nuclei possessed an average distance of 4.8 nm from DNA. This compares with a previously obtained distance in whole cells of 6.9 nm, for which 5 microM OP pretreatments decreased to 4.8 nm. These data indicate that tert-BuOOH generates DNA-damaging radicals through reaction with a subset of the different cell iron species which react with H2O2.

Depletion of cellular iron by bps and ascorbate: effect on toxicity of adriamycin.

A new method was developed that reduces the intracellular iron content of cells grown in serum-containing culture without involving the significant uptake of iron-chelating agents into cells. Negatively charged bathophenanthrolinedisulfonate (BPS), together with ascorbate, caused cells to lose much of their cellular iron without causing much depression in HL-60 or H9c2 (2-1) cell proliferation over a 48-h period. When added to serum supplemented RPMI-1640 culture media, BPS and ascorbate efficiently reduced and competed for iron in Fe(III) transferrin to form Fe(II)(BPS)3. The reaction also occurred with purified human iron-transferrin. When cells were incubated with growth medium containing serum that had been treated with BPS and ascorbate for 24 h, little or no BPS2- or Fe(II)(BPS)(4-)3 entered the cells, according to direct measurements and in agreement with the highly unfavorable 1-octanol/water partition coefficients for these molecules. However, iron was mobilized out of both cell types. After 24 h incubation of cells in this medium, there was no change in the activities of catalase and superoxide dismutase, or in the concentration of glutathione. Glutathione peroxidase was elevated 9%. Using HL-60 and H9c2 (2-1) cells made iron deficient with BPS and ascorbate, HL-60 cells grown in defined-growth media in the absence of iron-pyridoxal isonicotinoyl hydrazone, or Euglena gracilis cells maintained in a defined medium that was rigorously depleted of iron, it was shown that the cytotoxicity of adriamycin is markedly dependent on the presence of iron in each type of cell. Similar results were obtained when HL-60 cells were grown in RPMI-1640 culture medium and serum that had been incubated for 24 h in BPS and ascorbate and then chromatographed over a Bio-Rad desalting column to remove small molecules including BPS, ascorbate, and Fe(II)(BPS)3.

Evidence for involvement of multiple iron species in DNA single-strand scission by H2O2 in HL-60 cells.

Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsulfonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elution of DNA of cells treated at 4 degrees to minimize repair during treatment. Strand breakage in the presence of 10% serum was only 40% of that in the absence of serum. This effect was traced to reaction of H2O2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me2SO) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me2SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me2SO-inhibitable strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extracellular iron in serum was taken into account, 60 microM orthophenanthroline (OP) inhibited a maximum of 85% of strand breaks. In cells pretreated with 60 microM OP, the Me2SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decreased to 4.1 nm. These data indicate the existence of a number of different iron species, as characterized by overlapping but not coincidental inhibition by OP and Me2SO, and by differing diffusion distances.

Detection of myeloperoxidase gene expression in minimally differentiated acute myelogenous leukemia (AML-M0) using in situ hybridization.

Acute leukemias containing > 3% myeloperoxidase (MPO)-positive blast cells, as detected cytochemically, are considered to be myelogenous in origin, regardless of the immunophenotypic markers expressed. Conversely, acute leukemias that express only myeloid antigens are also considered to be acute myelogenous leukemia (AML), even in the absence of MPO. These MPO-negative AMLs, designated AML-M0 in the FAB classification, currently require either immunophenotypic or electron microscopic studies for identification. To examine the association of MPO and myeloid antigen expression in AML, particularly at the early stages of myeloid cell differentiation, we have used in situ hybridization (ISH) to evaluate MPO gene expression in myeloid leukemia cell lines and a variety of well-characterized acute leukemias, including six cases of AML-M0. Strong positivity for MPO mRNA was detected in the myeloid leukemia cell line HL-60 and in 22 of 27 AMLs (three AML-M0, four AML-M1, eight AML-M2, five AML-M4, two AML-M5a). No MPO gene expression was detected in three AML-M0, one AML-M5a, one AML-M7, 5 acute lymphoblastic leukemia, the lymphoid cell lines Molt-4 and Namalwa, or in the early myeloid cell lines KG-1 and KG-1a. Ultrastructural studies for MPO activity were performed on four AML-M0; one leukemia showed both gene expression and cytochemical activity, whereas two others contained neither MPO transcripts nor enzyme. Weak MPO gene expression was evident in one AML-M0 that was negative for enzymatic activity by electron microscopy. These studies show MPO gene expression can be detected by ISH in about half of AML-M0, supporting their presumed myelocytic derivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron requirement for cellular DNA damage and growth inhibition by hydrogen peroxide and bleomycin.

Studies with Euglena gracilis and HL-60 cells have assessed the need for intracellular iron in the mechanisms of inhibition of cell growth and DNA damage by H2O2 and bleomycin. Cell culture media were directly depleted of iron in order to deprive cells of nutrient iron. Major pools of cellular iron were reduced in both cell types. Nevertheless, iron bound in e.s.r.-observable haem protein and ribonucleotide diphosphate reductase in HL-60 cells was not decreased. In both control cell populations, there was a concentration-dependent reduction in proliferation and cell survival caused by H2O2. In comparison, the proliferation rates of both iron-deficient cell types were significantly less sensitive to H2O2. H2O2 caused concentration-dependent single-strand breakage in DNA in control HL-60 and Euglena gracilis cells. Iron deficiency reduced the amount of strand breaks in HL-60 cells at each concentration of H2O2 used. Single-strand breakage caused by H2O2 in Euglena gracilis was a direct function of the concentration of iron in which the cells had been grown. Growth inhibition and both single- and double-strand DNA damage caused by bleomycin were substantially reduced or eliminated in iron-deficient cells. Copper bleomycin behaved like metal-free bleomycin when assayed for the capacity to cause DNA damage in iron-normal and iron-deficient HL-60 cells. In contrast, iron bleomycin was equally active under the two conditions in these cells.

DNA strand breakage in isolated nuclei subjected to bleomycin or hydrogen peroxide.

The sources of iron (Fe) and reductant required for DNA strand breakage by the antitumor drug bleomycin (Blm), H2O2 and ascorbate were investigated using nuclei instead of whole cells in order to study a simpler, related system that was subject to better control and easier chemical manipulation. Ehrlich ascites tumor cells were isolated and treated directly on filters, and analysed for DNA damage by alkaline and nondenaturing elution. Extraction and treatment buffers were depleted of trace Fe by passage through Mg(OH)2 gel. Nuclei were treated for 1 hr at 37 degrees. High levels of single- and double-strand breakage were obtained using Fe(III)Blm in the range 0.01 to 0.08 microM. In contrast, Blm was effective only at two orders of magnitude greater concentration. Cu(II)Blm was totally ineffective in causing damage. Depletion of nuclear protein thiols with N-ethylmaleimide reduced double-strand breakage at the upper end of the FeBlm concentration-response curve. A 1 mM concentration of NADPH or NADH greatly increased the extent of double-strand breakage by 0.01 microM FeBlm, suggesting roles for cytochrome P450 or cytochrome b5 reductase in strand breakage. Fe(III)ATP (1:20 metal to ligand and 50 microM in Fe) and Fe(III)EDTA (1:2 metal to ligand and 50 microM in Fe) did not cause single-strand breaks. In the absence of added Fe, H2O2 or ascorbic acid (50 microM) caused less than one Gy-equivalent single-strand breakage. Addition of ascorbate plus Fe(III)ATP or Fe(III)EDTA produced breakage beyond the capacity of alkaline elution to analyse (5-6 Gy). Overall, the results indicate that Fe, which may contribute to DNA damage by Blm and forms of activated oxygen within cells, is not strongly bound in the nucleus and that nuclear thiols other than glutathione contribute reducing equivalents to Fe(III)Blm for the DNA damaging chemistry.

DNA double-strand breakage by bleomycin in Ehrlich ascites tumor cells as measured by nondenaturing filter elution.

DNA double-strand breakage by bleomycin (Bleo) in Ehrlich ascites tumor cells was examined by nondenaturing filter elution using 0.2% SDS at pH 9.6 as the eluant. The majority of damaged DNA from cells treated with 5 to 100 microM Bleo and for durations up to 6 h eluted in the first two or three 1.5-h fractions. This was significantly different from the multiphasic elution pattern of DNA from cells irradiated with 30 or 50 Gy 137Cs gamma rays, which showed a more gradual elution. Relative elution with respect to 50 Gy 137Cs gamma rays produced by 1-h treatments (about 0.35) showed no differences over the range of concentrations from 5 to 50 microM. Elution at pH 7.2 did not detectably reduce the fraction of DNA eluting from Bleo-treated cells, while reducing the elution of DNA from 50 Gy-irradiated cells by 35%. An experiment examining specificity for double-strand breakage by Bleo in different stages of the cell cycle demonstrated no differences in relative elution between G1, S and G2/M phases. Overall, the results of 1-h treatments are consistent with net production of high levels of double-strand breaks restricted to a portion of cellular DNA. However, extended treatments (up to 4 h) produced a nearly proportional increase in relative elution.

Repair of bleomycin-induced DNA double-strand breakage in Ehrlich ascites tumor cells.

The effect of 1,10-phenanthroline (OP) on repair of bleomycin (Bleo)-induced double-strand breaks in Ehrlich ascites tumor cells was studied using nondenaturing filter elution. 1,10-Phenanthroline is a metal chelator which is believed to inhibit strand breakage by Bleo through competition for intracellular iron. Cells were treated with 25 microM Bleo for 1 h, washed free of unincorporated drug, and then reincubated in the absence or presence of OP. In the absence of OP, relative elution (with respect to cells irradiated with 50 Gy and used as an internal standard) decreased in a first-order process with a half-time for repair of 2.4 h. 1,10-Phenanthroline at 10 nmol/10(5) cells (50 microM) accelerated the net decrease in relative elution, producing a biphasic response with half-times of 5.3 h and less than 30 min for the two components. Thus functionally active Bleo remained in cells after they were washed free of unincorporated drug. 1,10-Phenanthroline at a concentration of 3.1 nmol/10(5) cells did not result in a similar net acceleration of repair of double-strand breakage, though it increased the rate of repair of single-strand breakage as measured by alkaline elution. The differences in repair observed in response to different OP concentrations are discussed in terms of models for double-strand breakage by Bleo. After 4-5 h repair, relative elution from Bleo-treated cells remained at about 40% of that achieved at the end of 1-h Bleo treatment in either the presence or absence of the 10 nmol OP/10(5) cells, demonstrating that some double-strand breaks were resistant to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.

Properties of redox-inactivated bleomycins. In vitro DNA damage and inhibition of Ehrlich cell proliferation.

Blenoxane, bleomycin A2, bleomycin B2, and demethyl bleomycin A2 and products of their reactions with Fe2+ and oxygen were used to explore the relationship between their capacity to carry out in vitro DNA strand scission and their growth inhibitory activity against Ehrlich cells. Reaction of Fe2+, bleomycin and O2 in the absence of DNA decreased the subsequent effectiveness of various bleomycin congeners to degrade DNA in the presence of Fe2+ and oxygen. In comparison with controls, this loss of strand scission activity was not paralleled by equivalent decreases in growth inhibition. Demethyl bleomycin A2 retained full biological activity relative to bleomycin A2, despite being only 30% as effective as bleomycin A2 in its ability to cleave DNA in vitro. Prior reaction of bleomycins with Fe2+ did not alter their capacity to reduce oxygen or affect their ability to generate the activated intermediate which, for native bleomycin structures, is competent to cleave DNA in vitro.

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

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 microM 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 microM bleomycin, washed free of both drugs, and incubated at 35 degrees 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.

Spin-trapping studies of the oxidation-reduction reactions of iron bleomycin in the presence of thiols and buffer.

The reaction of ferrous bleomycin with dioxygen is reexamined to clarify whether radical species derived from molecular oxygen are generated. Detection of low levels of spin-trapped oxyradicals confirm the production of OH during this reaction when bleomycin is present in excess, but not when iron and drug concentrations are equal. In phosphate buffer, hydroxyl radicals continue to be spin trapped for at least 15 min after Fe(II)bleomycin has been oxidized to Fe(III)bleomycin. In HEPES buffer, detection of a HEPES radical in the absence of spin trap over the same period independently supports the conclusion that reactive radicals are present after the initial oxidation of Fe(II)bleomycin is complete. When glutathione is included in the aerobic reaction mixture, thiyl radical species are spin trapped. The reaction of Fe(III)bleomycin with cysteine produces thiyl radical without spin-trapped hydroxyl radical.