Novel fluorescent markers for hypoxic cells of naphthalimides with two heterocyclic side chains for bioreductive binding

Novel naphthalimides with two heterocyclic side chains of 2-nitroimidazole for bioreductive binding were designed, synthesized, and used as fluorescent markers for hypoxic cells. Their evaluation for imaging tumor hypoxia was carried out in V79 cells, CHO cells, and 95D cells in vitro by using fluorescence scan ascent. A2 and A4 showed a very large differential fluorescence between hypoxic and oxic cells (V79 cells) in vitro and are promising candidate markers for hypoxic cells.     

Synthesis and evaluation of novel 8-oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles as long-wavelength fluorescent markers for hypoxic cells in solid tumor

Novel bioreductive and long-wavelength fluorescent markers for hypoxic cells in solid tumor, 9-isocyano-8H-acenaphtho[l,2-b]pyrrol-8-one with the side chain of 2-nitroimidazole, were designed, synthesized, and evaluated in V79 379A Chinese hamster cells in vitro. Compounds A(2) and A(4) showed good hypoxic-oxic fluorescence differential in vitro (V79 cells) by using fluorescence scan ascent.     

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.     

Growth of V79 cells as xenograft tumors promotes multicellular resistance but does not increase spontaneous or radiation-induced mutant frequency

A Chinese hamster V79 xenograft model was developed to determine whether cells subjected to a hypoxic tumor microenvironment would be more likely to undergo mutation at the HPRT locus. V79-171b cells stably transfected with VEGF and EGFP were grown subcutaneously in immunodeficient NOD/ SCID mice. V79-VE tumors were characterized for host cell infiltration, doubling time, hypoxic fraction, vascular perfusion, and response to ionizing radiation. When irradiated in vitro, the mutant frequency for a given surviving fraction did not differ for cells grown in vivo or in vitro. Similar results were obtained using HCT116 human colorectal carcinoma cells grown as xenografts. However, V79-VE cells grown as xenografts were significantly more resistant to killing than monolayers. The background mutant frequency and the radiation-induced mutant frequency did not differ for tumor cells close to or distant from blood vessels. Similarly, tumor cells from well-perfused regions showed the same rate of strand break rejoining and the same rate of loss of phosphorylated histone H2AX as cells sorted from poorly perfused regions. Therefore, deleterious effects of the tumor microenvironment on DNA repair efficiency or mutation induction could not be demonstrated in these tumors. Rather, development of multicellular resistance in V79-VE tumors acted to reduce mutant frequency for a given dose of radiation.     

Radiosensitization of hypoxic cells by a nitrofuran; dose-modifying and shoulder effects.

The radiosensitizer nifurpipone dihydrochloride (5-nitro-2-furaldehyde N-methyl piperazino acetyl hydrazone dihydrochloride) sensitizes hypoxic V79 mammalian cells by at least two mechanisms. Sensitization is by a reduction of ? in addition to an increase in slope. Both these affects are absent under oxygenated conditions. When hypoxic V79 cells are irradiated in the presence of nifurpipone dihydrochloride combined with Ro-07-0582, sensitization greater than that due to air alone is observed; this effect is due to a reduction in ? and an increased slope. Again this effect is absent under oxygenated conditions. Rapid-mix studies using Serratia marcescens show that full senitization occurs with a pre-irradiation contact time of 4 msec; this contrasts with data for V79 cells where a pre-irradiation contact time of 40 msec is insufficient for any sensitization to occur. This sensitizer also exerts a differential toxic effect, being more toxic to hypoxic cells than to oxygenated ones. It is concluded from these results that nifurpipone dihydrochloride sensitizes by at least two mechanisms, one of which resembles that of the electron-affinic type.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Synthesis of a novel nitroimidazole-spermidine derivative as a tumor-targeted hypoxia-selective cytotoxin

A four-step synthesis of (R,S)-N(4)-[3-(2-nitro-1-imidazolyl)-2-hydroxypropyl]-spermidine trihydrochloride (4) is described and the utilization of the polyamine active transport system for the uptake of this compound in cells is demonstrated. Thus, V79 cells pretreated with an inhibitor of spermidine biosynthesis, alpha-difluoromethylornithine (DFMO), are ca. 2-fold more sensitive to 4 under hypoxic conditions, compared to untreated cells. Similarly, radiosensitization of hypoxic V79 cells by 4 is improved in DFMO-pretreated cells.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Comparison between the binding of [3H]misonidazole and AF-2 in Chinese hamster V79 spheroids

Binding of two hypoxia probes, [3H]misonidazole and AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide), was compared in Chinese hamster V79 spheroids incubated under different oxygen concentrations. Fluorescence-activated cell sorting based on Hoechst 33342 penetration was used to obtain populations of cells from different depths within the spheroid, and sorted cells were analyzed by cytofluorometry for AF-2 content and by liquid scintillation counting for [3H]misonidazole content. The patterns of AF-2 and misonidazole binding were very similar, with about 20-fold more localization of both drugs in anoxic compared to aerobic cells. Similar results were obtained when cells were sorted on the basis of AF-2 rather than Hoechst 33342 fluorescence. When mean cellular fluorescence of AF-2 was plotted versus cpm misonidazole/cell for different oxygen tensions, it appeared that oxygen was equally effective in inhibiting AF-2 and misonidazole binding. Internal cells of anoxic spheroids bound about twice as much AF-2 and misonidazole as external cells, apparently due to an increased rate of nitroreduction by chronically hypoxic cells. AF-2 was found to enhance the retention of misonidazole in oxic and hypoxic spheroids when both drugs were present.     

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.     

Radiosensitization of hypoxic mammalian cells in vitro by some 5-substituted-4-nitroimidazoles

The efficiencies of various 5-substituted-4-nitroimidazoles as radiation sensitizers have been determined in hypoxic Chinese hamster cells irradiated in vitro. Compared with published data on the sensitizing properties of substituted 2-nitro- and 5-nitroimidazoles, some of the 4-nitro derivatives show unusually high sensitizing efficiencies defined as the concentrations required to give an enhancement ratio of 1.6. The equilibrium one-electron reduction potentials of the compounds (E17) were measured by a pulse radiolysis technique and the results show that although sensitizing efficiencies are unexpectedly high, based on considerations of electron affinity, they still increase with increasing values of E17. Enhancement ratios were determined in two V79 cell lines for combinations of one of these compounds (a 4-nitroimidazole containing the group SO2.O.phenyl in the 5-position, NSC 38087) with various concentrations of misonidazole. The sensitization observed suggests that the two compounds may be operating by different mechanisms.     

Electron-Affinic Sensitization VII. A Correlation between Structures, One-Electron Reduction Potentials, and Efficiencies of Nitroimidazoles as Hypoxic Cell Radiosensitizers

Radiosensitization efficiencies for seven different 2-nitroimidazoles including Ro-07-0582 and its urinary metabolite, Ro-05-9963, and two 5-nitroimidazoles including metronidazole, have been determined in hypoxic Chinese Hamster cells, line V79-379A, X-irradiated in vitro. All the compounds were active hypoxic cell sensitizers with the enhancement ratios increasing with drug concentration. The 2-nitroimidazoles were all more efficient than the 5-nitroimidazoles. Overall, the efficiencies, defined as the concentration required to give a particular enhancement ratio, varied by a factor of about 200. Electron-affinities of the sensitizers were determined by pulse radiolysis as the one-electron reduction potentials and these correlate well with the sensitization efficiencies of the compounds. The correlation extends beyond the nitroimidazole series as is shown by data for p-nitroacetophenone, nifuroxime (a nitrofuran) and oxygen itself. The nitroimidazoles varied by a factor of 70 in their octanol/water partition coefficients, but the effect of this parameter on sensitizing efficiency is small compared with the influence of electron affinity.     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4-6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC(50)=80 microM compared with 17 microM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC(50)=4 micro compared with l microM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC(50) of 380 microM in V79-GSTA5 compared with 180 microM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal-glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.     

Design of a bioreductively-activated fluorescent pH probe for tumor hypoxia imaging

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals

We recently constructed a Chinese hamster V79-derived cell line that stably expresses human cytochrome P450 (CYP) 2E1 and human sulphotransferase (SULT) 1A1. These enzymes are involved in the bioactivation of numerous promutagens/procarcinogens, but are not taken into account in standard in vitro mutagenicity assays. Various carbohydrate pyrolysis products and other food contaminants that induce tumours or preneoplastic lesions in laboratory animals are inactive or only weakly active in standard in vitro genotoxicity assays. This is the case for acrylamide, furan, 5-hydroxymethylfurfural, nitrofen and N-nitrosodimethylamine. These compounds were investigated for induction of sister chromatid exchange (SCE) in V79-hCYP2E1-hSULT1A1 cells. All test compounds showed positive results over a wide concentration range, starting at 0.01 microM for N-nitrosodimethylamine, 3 microM for furan, 12.5 microM for nitrofen, 20 microM for 5-hydroxymethylfurfural, and 200 microM for acrylamide. The concentration-response curve of furan was unusual, as this compound induced a statistically significant, but rather constant and weak increase in SCE over an extremely wide concentration range (3-16,000 microM). Furan was slightly less active, whereas the remaining compounds were much less active in the parental V79 cell line than in V79-hCYP2E1-hSULT1A1 cells. Compared to many other genotoxic effects, the study of SCE only requires small numbers of cells (and incubation volumes) and usually is detected even at low concentrations of the genotoxicant. Therefore, induction of SCE in V79-hCYP2E1-hSULT1A1 cells may be useful in the genotoxicity testing of preparations of heated food and in their bioassay-directed fractionation.     

Abnormal radiosensitizing and cytotoxic properties of ortho-substituted nitroimidazoles

Various 5-substituted 4-nitroimidazoles have been shown to be much more efficient radiosensitizers and much more toxic than would have been predicted from their electron affinities, as measured by values of one-electron reduction potential, E17. Using Chinese hamster V79 cells in vitro, a comparison has been made with some isomeric 4-substituted 5-nitroimidazoles. These compounds have E17 values some 64mV greater than the 4-nitroimidazoles, yet show much lower sensitizing efficiency and also lower toxicity. Neither series of compounds shows the greater toxicity towards hypoxic cells usually associated with nitroaromatic and nitroheterocyclic compounds. The second-order rate constants, k2, for reaction of these isomeric nitroimidazoles with glutathione and dithiothreitol were determined. Within each series the value of k2 increased with increasing electron affinity, however, the 4-nitroimidazoles were always more reactive than their corresponding 5-nitro isomers. The sensitizing and toxic properties of these compounds may involve depletion of intracellular thiols; this possibility is discussed.     

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.