Norma J. Nelson

Characterization of Radiolabeled Fluoromisonidazole as a Probe for Hypoxic Cells

Radiolabeled fluoromisonidazole has been characterized as a probe for hypoxic cells in vitro and in vivo. The uptake and retention of [3H]fluoromisonidazole and [3H]misonidazole were compared in V-79 cell monolayers and spheroids by varying incubation time and O2 levels in contact with the medium. The two labeled drugs were retained similarly in cell populations isolated from different depths in spheroids, and the amount of each drug bound in cells at the spheroid periphery increased with decreasing O2 level. The labeling patterns in autoradiographs were similar for spheroids incubated with the two labeled drugs, with most silver grains located over a zone of viable and presumed hypoxic cells intermediate between the necrotic center and the periphery of the spheroid. Biodistribution of the two tritiated drugs was compared in C3H mice bearing KHT tumors with 15% radiobiologically hypoxic cells. Tumor:blood and tumor:muscle ratios greater than 5.0 were achieved in mice sacrificed 4 h after the last of three injections of 5 or 20 mumol/kg of [3H]fluoromisonidazole. These ratios are compatible with imaging and are higher than those obtained with 50 mumol/kg misonidazole in a similar administration protocol. TLC analysis of plasma from mice injected with [3H]fluoromisonidazole indicated that the drug was stable in vivo for up to 2 h and that the metabolites formed were too polar to be dehalogenation products. Fluoromisonidazole labeled with 18F at the end of the alkyl side chain would retain the label on metabolites that bind in hypoxic cells in vivo. Fluoromisonidazole binds stably in the same populations of hypoxic cells as does misonidazole, and we conclude that [18F]fluromisonidazole has potential use as a hypoxia imaging agent in vivo.

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution.

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.

Discrepancies between Patterns of Potentially Lethal Damage Repair in the RIF-1 Tumor System in Vitro and in Vivo'

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.

Repair of Potentially Lethal Damage Following Irradiation with X Rays or Cyclotron Neutrons: Response of the EMT-6/UW Tumor System Treated under Various Growth Conditions in Vitro and in Vivo

Postirradiation potentially lethal damage (PLD) repair was examined in the EMT-6/UW tumor system under a variety of in vitro and in vivo growth conditions. Following x irradiation, surviving fraction increased in fed and unfed plateau cultures if subculture and plating were delayed; in exponentially growing cultures if they were covered with depleted medium for the first 6 h postirradiation; and in tumors in vivo if excision for preparation of a cell suspension was delayed. Following irradiation with 21.5 meV (d/sup +/ ..-->.. Be) neutrons, PLD repair was measurable only in unfed plateau cultures when subculture was delayed and in exponentially growing cells exposed to depleted culture medium immediately after irradiation. In x-irradiated EMT-6/UW cells, the greatest repair capacity and the highest surviving fraction ratios were measured in unfed plateau cultures; the least repair was observed in exponentially growing cells exposed to depleted medium. Thus post-neutron repair was not limited to situations where the amount of repair of photon PLD is large. The demonstration of PLD repair in tumors irradiated in vivo with X rays and the absence of such repair after neutrons could have important implications in radiotherapy if this is a general phenomenon.

Comparison of binding of [3H]misonidazole and [14C]misonidazole in multicell spheroids.

Uptake of [2-ring-14C]misonidazole and [3H]misonidazole with tritium in the side chain has been compared in 1-mm EMT-6/UW spheroids using liquid scintillation counting and autoradiography. The uptake of both labeled sensitizers as a function of incubation time was virtually identical. Uptake by the spheroids exceeded levels in the medium by 11/2 to 2 hr and was well modeled as a first-order binding process, with rate constants of 0.00324 hr-1 for 3H and 0.00388 hr-1 for 14C. The similar uptake of the two versions of this sensitizer labeled in different positions suggests that the metabolic actions which allow the drug to bind in hypoxic cells do not principally involve metabolites which separate the number 2 carbon of the imidazole ring from the side chain. The pattern of silver grains in autoradiographs was similar for both labeled sensitizers, with most labeled drug bound in an intermediate zone of cells between the necrotic center and the actively proliferating rim of the spheroids. The superior resolution possible with the tritiated compound showed that both nucleus and cytoplasm in viable looking cells were labeled while pycnotic cells were not labeled.

Cure of EMT-6 Tumors by X Rays or Neutrons: Effect of Mixed-Fractionation Schemes

EMT-6 mouse tumors were treated with (a) 1 or 5 fractions of either x rays or neutrons or (b) a mixture of both in which 2 fractions of neutrons (n) plus 3 fraction of x rays (x) were given in 5 days in the sequence n-n-x-x-x or n-x-x-x-n. Using local tumor control as an end point, neutron RBEs of 1.7 for single fractions and 2.6 for 5 fractions were obtained. The two mixed schemes yielded RBEs of 1.5 and 1.4, respectively. Therapeutic gain factors, calculated as the ratio of tumor to skin RBE with neutrons or mixed radiations, were highest for the mixed fractionation schemes. These results are due to an apparent enhancement of the neutron effect in the tumor but not in the skin with these regimens. Other normal tissues must be irradiated with mixed schemes to determine whether this phenomenon is limited to the skin.

Relationship of iron metabolism to tumor cell toxicity of stable gallium salts

Abstract The antitumor properties of citrated gallium nitrate (NSC 15200) have been tested using the EMT-6/UW mouse sarcoma growing in vitro or in vivo . This tumor system has been used in both growth forms in our studies which led to the transferrin receptor hypothesis of the mechanism of uptake of tracer levels of gallium-67 citrate. Gallium nitrate is both cytostatic and lethal in vitro with some growth inhibition occurring after chronic exposure to low doses (10 μg per ml) which kill essentially no cells. Cell kill and growth inhibition were both observed if cells were exposed for longer times at higher doses. The effects of gallium nitrate were enhanced by the addition of human transferrin to the culture medium. The enhanced toxicity was consistent with, and proportional to, increased gallium uptake in the presence of transferrin. The addition of ferric citrate greatly reduced the toxic effect of the gallium salt. The enhancing effect of transferrin and protective action of ferric citrate are consistent with our studies of the effect of these two agents on concentration of tracer levels of the tumor-imaging radiopharmaceutical, carrier-free gallium-67 citrate. Tumors in vivo were largely resistant to 10 daily doses of 90 mg kg −1 of gallium nitrate. Ga may mimic Fe in some aspects of cell metabolism. Competition between the two metals occurs at the initial uptake step (binding to transferrin) and possibly to a lesser extent at other points in cellular incorporation and deposition. Iron has been implicated as a regulatory factor in cell growth and may be essential for cell proliferation. The toxicity of stable gallium is interpreted in the context of similarities of metabolism of the two metals.

Toxicity, biodistribution and radioprotective capacity of l-homocysteine thiolactone in CNS tissues and tumors in rodents: comparison with prior results with phosphorothioates

L-Homocysteine thiolactone (L-HCTL) was evaluated for its potential as an intravenously-administered central nervous system (CNS) radioprotector in C3H mice and F344 rats. Toxicity assessments in the mouse yielded a LD50 of 297 mg/kg and in the rat 389 mg/kg. Biodistribution studies in tumor-bearing mice showed that brain specimens contained more label at 10 min than the tumors but less at 30 or 60 min. Brain uptake relative to the tumors, the brain/tumor ratio, ranged between 0.5 and 3.3. The cervical spinal cord of non-tumor-bearing rats was irradiated with 32 Gy 137Cs with or without prior treatment with L-HCTL following which the time to forelimb or hindlimb paralysis was measured to determine the relative protective factors (RPFs) for this radiation dose. For forelimb paralysis the RPF was 1.9 (+/- 1.0, SD) and for hindlimb it was 2.0 (+/- 1.1, SD). 36B-10 glioma cells irradiated in vitro with or without L-HCTL and assayed for colony forming capacity demonstrated a dose modifying factor (DMF) of only 1.15 (+/- 0.16, SE). Rats bearing intracerebral 36B-10 glioma received 137Cs irradiation with or without L-HCTL after which the tumors were similarly assayed in vitro. From this the glioma DMF was 1.2 (+/- 0.30, SE). Compared to prior results with phosphorothioates our data show that the toxicity of L-HCTL is roughly the same as WR2721, WR77913 and WR3689 and that it distributes at higher levels in the CNS after systemic administration. L-HCTL may well equal these phosphorothioates at protecting normal CNS tissue without requiring administration directly into the cerebrospinal fluid-containing spaces and it does not protect the 36B-10 glioma.

Effect of tumor dissaggregation on results of in vitro cell survival assay after in vivo treatment of the EMT-6 tumor: X-rays, cyclophosphamide, and bleomycin

SummaryEMT-6 tumors were treated in vivo with 300 kVp X-rays, cyclophosphamide, or bleomycin. Tumor cell suspensions were prepared by digesting tumors with trypsin or a collagenase-deoxyribonuclease-pronase cocktail, and cells were plated in vitro for determination of fractional cell survival. Cell survival after X-rays was identical for the two disaggregation methods. Trypsin-derived cells were far more sensitive to bleomycin but less sensitive to cyclophosphamide than those prepared with the mixed enzyme cocktail. Interaction of drug produced and enzyme caused damage was the probable cause for these discrepancies. The nature of the interaction may be drug specific and therefore unpredictable. The results were unlikely to be due to different nonrepresentative tumor cell samples being produced by the two digestion methods, because the X-ray cell survival curves were so similar for the two products.

Recovery from potentially lethal damage following irradiation with x-rays or cyclotron neutrons—I Response of emt-6 cells in vitrot

Abstract EMT-6 cells in vitro , in a nutritionally limited plateau stage of growth, were used to study recovery from potentially lethal radiation damage. H subculture was delayed after irradiation with X-rays or cyclotron neutrons, recovery from potentially lethal damage was seen as an increase in survival relative to that in cells subcultured immediately after irradiation. The time course of damage repair seemed to be similar after both types of radiation; the extent of recovery, while variable, can be large after the high linear energy transfer (LET) exposure as well as after photon radiation. When parallel X-ray and neutron experiments were performed with the same culture passage, the amount of post-neutron repair was less than that occurring after X-irradiation.