J. D. Chapman

The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques.This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

The recently obtained low value of approximately 1.5 for the alpha/beta of prostate cancer has led us to reexamine the optimal prostate tumor biology parameters, while taking into account everything known about the radiation response of prostate clonogens for use in a predictive dose-response model.Averages of the literature values of the alpha- and beta-inactivation coefficients for human prostate cancer cell lines were calculated. A robust tumor local control probability (TLCP) model was used that required average alpha and beta, as well as sigma(alpha), for the interpatient variation in single-hit killing (alpha). Median PO(2) values <or=1 mm Hg in the prostates of Fox Chase Cancer Center brachytherapy patients had been found in 21% of 115 cases. The oxygen enhancement ratios of 1.75 and 3.25 for alpha- and beta-inactivation, respectively, measured for tumor cells in vitro, were incorporated into the TLCP model, together with a clonogen density of approximately 10(5) cells/cm(3). Severe hypoxia and radioresistance were estimated for a proportion of tumors that was increased with PSA level.For asynchronous human prostate cell lines irradiated in air, alpha(mean) was 0.26 +/- 0.07 (standard error) Gy(-1), sigma(alpha) = 0.06 Gy(-1), and beta(mean) was 0.0312 Gy(-2) +/- 0.0064 (standard error) Gy(-2). The TLCP data indicated that most tumors that contained aerobic cells would be cured, whereas most tumors that contained hypoxic cells would not be cured by total doses of 76 to 80 Gy. Clinical response data from the literature for external beam dose escalation, stratified by PSA value, and for low-dose-rate brachytherapy, were well predicted by the model, where the alpha/beta ratio was 8.5 and 15.5 for well-oxygenated and hypoxic clonogens, respectively.Neither alpha/beta ratio nor clonogen number need be extremely low to explain the response of prostate cancer to brachytherapy and external beam therapy, contradicting other recent analyses. It is strongly suggested that severe hypoxia in the prostates of certain patients limits the overall cancer cure rate by conventional radiation therapy.

To investigate whether low partial pressure of oxygen (PO2) in prostate cancer (CaP) predicts for biochemical outcome after radiotherapy. We previously reported that hypoxic regions exist in human CaP.Custom-made Eppendorf PO2 microelectrodes were used to obtain approximately 100 PO2 readings from both pathologically involved regions of the prostate (as determined by sextant biopsies) and normal muscle (as an internal control). Fifty-seven patients with localized disease were prospectively studied; all received brachytherapy implants (48 low dose rate and 9 high dose rate) under spinal anesthesia. Nine patients had received prior hormonal therapy. Biochemical failure was defined as two consecutive rises in prostate-specific antigen level, without a return to baseline. Cox proportional hazards regression analysis was used to evaluate the influence of hypoxia on biochemical control, while adjusting for prostate-specific antigen, Gleason score, stage, implant type (low dose rate versus high dose rate), perineural invasion, hemoglobin level, use of hormonal therapy, average (mean) of the median prostate PO2, average median muscle PO2, and prostate/muscle PO2 (P/M) ratio.With a median follow-up of 19 months (range 4 to 31), 9 patients developed biochemical failure. A threshold analysis of the P/M ratio demonstrated that biochemical control at 2 years differed significantly at a ratio of less than 0.05 versus 0.05 or greater (31% versus 92%, P <0.0001). However, the classic prognosticators were similar in these two groups. On multivariate analysis, the P/M ratio was the only predictor of biochemical control (P = 0.0002).To our knowledge, this is the first study to correlate the degree of hypoxia in CaP with treatment outcome after radiotherapy. The P/M PO2 ratio was the strongest predictor for biochemical control on stepwise multivariate analysis. Longer follow up with more patients is planned to confirm this result.

The yields of unrepairable single-and double-strand breaks in the DNA of x-irradiated Chinese hamster cells were measured by low-speed neutral and alkaline sucrose density gradient sedimentation in order to investigate the relation between these lesions and reproductive death. After maximal single-strand remoining, at all doses, the number of residual single-strand breaks was twice the number of residual double-strand breaks. Both double-strand and unrepairable single-strand breaks were proportional to the square of absorbed dose, in the range 10-50 krad. No rejoining of double-strand breaks was observed. These observations suggest that, in mammalian cells, most double-strand breaks are not repairable, while all single-strand breaks are repaired except those that are sufficiently close on complementary strands to constitute double-strand breaks. Comparison with cell survival measurements at much lower doses suggests that loss of reproductive capacity corresponds to induction of approximately one double-strand break.

In order to demonstrate whether “spontaneous” erythroid colonies observed in vitro in polycythemia vera (PV) using standard colony assays were independent from erythropoietin (epo) or exquisitely sensitive to the hormone, we used methyl cellulose serum-free cultures, in which serum was completely replaced by iron-saturated transferrin, α-thioglycerol albumin, and low density lipoproteins. Connaught Step III epo was used. In 6 cases of PV, no epo-independent colony (CFU-E or BFU-E derived) was observed in serum-free conditions, while spontaneous colonies were present after plating the same PV bone marrow in culture with serum. The epo dose-response curves showed a tenfold increase in the sensitivity to epo of PV erythroid progenitors compared to normal controls. In PV, the first CFU-E and BFU-E colonies were observed after addition of 0.001–0.01 lU/ml of epo, while in controls they appeared at an epo concentration between 0.01 and 0.1 lU/ml. Numbers of spontaneous colonies in cultures with serum compared with the epo dose-response curve in the same patient in serum-free cultures are much higher than expected from the small amount of epo present in the serum. These results confirm that PV erythroid progenitors able to differentiate spontaneously in standard culture conditions are in fact dependent of epo and hypersensitive to the hormone. They show that these abnormal progenitors do not represent a homogeneous population, but exhibit different degrees in their hypersensitivity to epo. Since the small amount of epo present in the normal serum cannot explain the growth of spontaneous colonies by itself, a hypersensitivity to other serum factors cannot be excluded.

Objectives. The purpose of this study was to characterize, by use of the Eppendorf microelectrode, the extent of hypoxia (range/heterogeneity) in human prostate carcinomas. Methods. Custom-made Eppendorf pO2 microelectrodes were used to obtain pO2 measurements from the pathologically involved side of the prostate, as well as from a region of normal muscle for comparison. Each set of measurements comprised approximately 100 separate readings of pO2, for a total of 2145 individual measurements. Twelve patients were studied, 7 of whom underwent brachytherapy, 3 a radical prostatectomy, and 2 a cystoprostatectomy. The pO2 measurements were obtained in the operating room, using sterile technique, under spinal anesthesia for the brachytherapy group patients and under general anesthesia for the surgery group patients. The Eppendorf histograms were recorded and described by the median pO2, mean pO2, and percentage of measurements less than 5 mm Hg and less than 10 mm Hg. Results. Because of differences in patient characteristics and the anesthesia employed, control measurements were obtained from nearby normal muscle as an internal control in all but 2 patients. This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower than those from normal muscle. Similarly, higher pO2 readings were obtained from the pathologically normal prostates (in the patients with bladder cancer) than from the prostates of patients with prostate carcinoma. Increasing levels of hypoxia were observed with increasing clinical stage. Significant predictors of oxygenation include the type of tissue (pathologically involved prostate versus normal muscle or normal prostate), clinical stage, and type of anesthesia. Conclusions. This report, to our knowledge, represents the first study to obtain in vivo electrode measurements of oxygen levels in patients with prostate cancer and suggests that hypoxic regions exist in human prostate carcinoma. More patients will be accrued to this prospective study to correlate the oxygenation status of prostate carcinoma with known prognostic factors and treatment outcome.

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

[14C]Misonidazole (MISO) becomes bound to macromolecules of mammalian cells upon hypoxic incubation. Intracellular enzyme processes are implicated since the temperature dependence for this process showed an activation energy of 33.5 kcal/mol. The sensitizer bound to both hypoxic and aerobic cells was associated with the macromolecular fraction and the soluble fraction in the proportion, 23 and 77%, respectively. The initial rate of binding of [14C]MISO to the macromolecular (acid-insoluble) fraction of hypoxic EMT-6 mouse tumor and V-79 hamster cells increased proportionally with the square root of extracellular concentration of MISO up to at least 5mM. High concentrations of dimethyl sulfoxide (an effective OH radical scavenger), allopurinol (an effective inhibitor of xanthine oxidase), and diamide (a chemical which can deplete cellular levels of glutathione) had little or no effect on this metabolism-induced binding process. The addition of high concentrations of exogenous cysteamine to hypoxic cell cultures resulted in almost complete inhibition of binding. Extracellular bovine albumin at high concentration in hypoxic cell cultures had little effect on the production of adducts to cell macromolecules and only small amounts of [14C]MISO were found to bind to the extra-cellular bovine albumin. This result suggests that MISO preferentially binds to molecules within the cell in which it is metabolically activated. In experiments where cells labeled under hypoxic conditions with [14C]MISO were subsequently permitted to proliferate in aerobic monolayers, a half-life of the acid-insoluble addition products of approximately 55 hr was measured. A large number of [14C]MISO adducts (approximately 10(9)/cell) can be generated in hypoxic cells without any evidence of cytotoxicity, and they are slowly cleared from cells. These are favorable characteristics as regards the development of this technique as a marker for hypoxic cells in solid tumors.

The fluorometric technique for measuring the levels of reduced pyridine nucleotides was used to study oxidative metabolism in isolated rabbit papillary muscle at 23 degrees C. The 100% standard level of tissue fluorescence was defined as that measured for muscles resting in oxygenated 10 mM pyruvate solution. This level increased 15% with anoxia and decreased 45% with stimulation in substrate-free solution. Thus, about one-half of the standard tissue fluorescence was metabolically labile and this labile fraction is suggested to be mitochondrial in origin. Decreased tissue fluorescence following mechanical activity was identified with increased oxidation of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) owing to stimulation by adenosine diphosphate (ADP), released during activity, of mitochondrial respiration. The kinetics of the fluorescence transients were slowed fourfold by removal of pyruvate. This effect was not significantly reversed by addition of 10 mM glucose. The time integrals of the fluorescence transients were linearly related to the amounts of mechanical activity in the presence, but not in the absence, of pyruvate. A positive correlation was observed between the steady-state peak tension at constant stimulus rate and the resting level of reduction of pyridine nucleotides in various media. The fluorometric results are interpreted to be indicative of the steady and transient states established by the substrate dehydrogenases and the respiratory chain during oxidative phosphorylation in mitochondria.

Various nitrofuran derivatives have been tested with Chinese hamster cells growing in vitro to determine their toxicity and radiosensitizing effectiveness. All nitrofurans tested displayed a greater radiosensitizing potential than p-nitroacetophenone for hypoxic cells at concentrations producing no acute toxicity. The most effective compound tested to date is nifuraldezone which at 250 μm in complete medium gives the same extent of radiosensitization (ER∼2·85) as oxygen in air-saturated medium. Additional nitroheterocyclic compounds in current clinical usage have also been characterized. The chemical and pharmacological properties of the various compounds are discussed in relation to their application in animal tumour studies. High water solubility appears to be an important property for radiosensitizers to be used in animal tumour studies. A positive demonstration of radiosensitization in vivo with nitroheterocyclic drugs will depend upon whether or not a sensitizing concentration of the drugs can be established in the hypoxic cells of a solid animal tumour for the duration of the radiation treatment.

The radiation inactivation of the proliferative capacity of Chinese hamster fibroblasts is described by the linear--quadratic relationship, S/S$sub 0$ = exp (-$alpha$D - $beta$D$sup 2$). Values of $alpha$ and $beta$ have been determined for asynchronous cells irradiated under various conditions of chemical radioprotection and radiosensitization. OH has been found to contribute to cell inactivation by both $alpha$ (single-events) and $beta$ (double-events). Sulfhydryl repair was found to provide radioprotection against single- and double- events while molecular oxygen was found to radiosensitize predominantly by enhancing double-events. Electron-affinic radiosensitizers enhanced the double- event component. These radiation chemical effects on cell inactivation are discussed with reference to potential molecular targets and lesions and the microdosimetry of 250 kV X rays in water. (auth)

Reovirus virions, grown in suspension cultures of L cells and extensively purified by density gradient and velocity gradient centrifugation after their release from cell debris by fluorocarbon extraction, are characterized by a mean particle diameter of 73 nm and a density in CsCl of 1.36 to 1.37 g/cm(3). Treatment of intact virions by chymotrypsin (CHT) digestion in vitro converts them to subviral particles (SVP) having characteristics which are determined by the species of monovalent cation present during the digestion. In the presence of Cs(+) ions, CHT converts the virions to SVP of mean diameter 51 nm and density 1.43 to 1.44 g/cm(3). In the presence of K(+) ions, the conversion is to SVP of diameter 51 nm and density 1.39 to 1.40 g/cm(3). The SVP made in the presence of either Cs(+) or K(+) possess an extremely active RNA polymerase and nucleoside triphosphate phosphohydrolase (NTPase) activity in vitro and are resistant to further digestion by CHT. Treatment of intact virions with CHT in the presence of Na(+) or Li(+) ions results in their conversion to SVP of mean diameter 64 nm and density 1.37 to 1.38 g/cm(3). Such SVP are not active in in vitro RNA synthesis or NTP hydrolysis and are resistant to further digestion by CHT even during prolonged exposure to high concentrations of enzyme. Addition of Cs(+) or K(+) ions to the digestion mixture allows conversion of the 64-nm diameter SVP to 51-nm diameter SVP in which the RNA polymerase and NTPase are active in vitro. Analysis of the proteins present in intact virions and in the different SVP reveals clear differences which indicate that the conversions are accomplished by removal or cleavage of particular species of polypeptides.

Reproductive survival of irradiated Chinese hamster fibroblasts is adequately described at all cell ages by an equation of the form $S/S_{0}={\rm exp}(-\alpha D-\beta D^{2})$. The two terms may be related to the likelihood of inactivation by one or two events, respectively. If the inactivating lesion involves damage to the genome, then corrections should be applied to all but G1 phase cell populations to account for genome multiplicity. The appropriate corrections are derived. The corrections render the single event coefficient, a, constant through interphase, in agreement with data for high LET irradiation. The variations in the two parameters with cell age are clearly different, indicating that cyclic changes in radiosensitivity may not be related to any single physiological correlate, and suggesting that there may be mechanistic differences between the two components other than the number of events involved. Synthesized survival data representing an exponential population are well fitted by the same eq...

A nucleoside triphosphate phosphohydrolase activity has been discovered in reovirus virions. This activity converts nucleoside triphosphates to nucleoside diphosphates in vitro. Properties of this enzyme are presented, with evidence that this activity is an integral part of the virion core.

(1974). Studies on the Radiosensitizing Effect of Oxygen in Chinese Hamster Cells. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine: Vol. 26, No. 4, pp. 383-389.

The purpose of this study was to characterize the extent of hypoxia in human prostate carcinoma using the Eppendorf PO2 microelectrode. Custom-made Eppendorf PO2 microelectrodes were used to obtain PO2 measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies), as well as from a region of normal muscle for comparison. Fifty-nine patients with localized prostate cancer were studied, all of whom received brachytherapy implants under spinal anesthesia. A multivariate mixed effects analysis for prediction of tumor oxygenation was performed including the following covariates: type of tissue (prostate versus muscle), prostatic-specific antigen, disease stage, patient age and race, tumor grade, volume, perineural invasion, and hormonal therapy. Because of differences in patient characteristics, control measurements were obtained from normal muscle in all patients. This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median PO2 = 2.4 mm Hg) compared with the measurements from normal muscle (average median PO2 = 30.0 mm Hg), p < 0.0001. A multivariate, linear, mixed analysis demonstrated that the only significant predictor of oxygenation was the type of tissue (prostate versus muscle). This study, using in vivo electrode oxygen measurements, suggests that hypoxia exists in human prostate carcinoma. More patients will be accrued to this study to ultimately correlate the oxygenation status in prostate carcinoma tumors with treatment outcome.

Asynchronous populations of mouse EMT-6 tumor cells were treated with Photofrin II and exposed to various doses of 630 nm light in slowly stirred suspensions which had been equilibrated with various concentrations of oxygen. Survival curves were generated with cells exposed to 20 micrograms/ml Photofrin II in tissue culture medium for 1 h, a procedure which made it possible to remove more than 50% of the drug by washing. It is expected that under these conditions the drug would be loosely bound to cell surface and plasma membranes and in the cellular cytosol. Survival curves were also generated with cells exposed to 5 micrograms/ml Photofrin II for 20-24 h, a procedure which resulted in greater than 90% of the drug being tightly bound within cells, presumably to cellular lipids and membranes. Oxygen was obligatory for killing cells which had been exposed for both "short term" and "long term" to Photofrin II. After 30-40 min of pregassing cells with nitrogen gas which contained precise levels of oxygen, the concentration required to reduce rates of cell killing to 50% of maximum was approximately 0.5% O2 (gas phase) for short-term drug exposures and less than or equal to 0.05% O2 for long-term drug exposures. Even after pregassing times of 90-120 min prior to light administration, a Km of approximately equal to 0.1% O2 was observed for cells exposed to the drug for the longer time. When the same cells were exposed to 137Cs gamma rays in this irradiation chamber, no change in radiation sensitivity was observed after 30 min of pregassing cells with all oxygen concentrations studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to analyze the extent of hypoxia in prostate carcinoma tumors using the Eppendorf pO(2) microelectrode and correlate this with pretreatment characteristics and prognostic factors.Custom-made Eppendorf pO(2) microelectrodes were used to obtain pO(2) measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies) as well as from a region of normal muscle for comparison. Each set of measurements comprised approximately 100 separate readings of pO(2), for a total of 10,804 individual measurements. Fifty-five patients with localized prostate carcinoma were studied: Forty-one patients received brachytherapy implants, and 14 patients underwent radical prostatectomy. The pO(2) measurements were obtained in the operating room by using a sterile technique under spinal anesthesia for the brachytherapy group and under general anesthesia for the surgery group. The Eppendorf histograms were recorded and described by the median pO(2), mean pO(2), and percentage < 5 mm Hg and < 10 mm Hg. A multivariate mixed-effects analysis for the prediction of tumor oxygenation was performed and included the following covariates: type of tissue (prostate vs. muscle), type of treatment (implant vs. surgery) and/or anesthesia (spinal vs. general), prostate specific antigen level, disease stage, patient age and race, tumor grade, tumor volume, perineural invasion, and hormonal therapy.Due to differences in patient characteristics and the anesthesia employed, control measurements were obtained from normal muscle (in all but two patients). This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median pO(2), 9.9 mm Hg) compared with the measurements normal muscle (average median pO(2), 28.6 mm Hg; P < 0.0001). A multivariate, linear, mixed analysis demonstrated that, among all of the patients, the significant predictors of oxygenation were tissue (prostate vs. muscle) and anesthesia (spinal vs. general) or treatment (implant vs. surgery). Among the brachytherapy (spinal anesthesia) patients, the significant predictors of pO(2) were tissue type, disease stage, and patient age. There were no significant predictors of oxygenation in the surgical (general anesthesia) group.This study, employing in vivo electrode oxygen measurements, demonstrated that hypoxia exists in prostate carcinoma tumors. A dramatic effect of anesthesia was observed, likely due to modulation of polarography in the presence of fluorine. Within the group of brachytherapy (spinal anesthesia) patients, increasing levels of hypoxia (within prostatic tissue) correlated significantly with increasing clinical stage and patient age. More patients will be accrued to this prospective study to further correlate the oxygenation status in prostate carcinoma tumors with known prognostic factors and, ultimately, treatment outcome.

<h3>Background:</h3> The treatment of patients with gallstones who have suffered a first episode of acute biliary pain is controversial. Recent guidelines suggest that such patients may choose to observe the "pattern" of their pain over time before deciding about therapy. <h3>Objective:</h3> To determine clinical factors that would identify patients at high risk for 2 important complications: acute biliary pancreatitis and acute cholecystitis. <h3>Methods:</h3> We collected sociodemographic and clinical data on patients undergoing cholecystectomy after acute biliary pancreatitis, acute cholecystitis, or uncomplicated biliary pain. The physical characteristics of gallstones recovered at surgery were also recorded. Patients with pancreatitis and patients with cholecystitis were compared with patients with uncomplicated pain. <h3>Results:</h3> In univariate analyses, patients with acute pancreatitis were significantly more likely to have at least 1 gallstone smaller than 5 mm in diameter, 20 or more gallstones, gallstones described as mulberry shaped, and a lower total gallstone weight than patients with uncomplicated pain. Pancreatitis was unrelated to patient age, sex, race or ethnicity, use of alcohol or tobacco, or clinical comorbidity. In a logistic regression model, acute pancreatitis was associated with a stone diameter of less than 5 mm (odds ratio, 4.51;<i>P</i>=.007) and with mulberry-shaped gallstones (odds ratio, 2.25;<i>P</i>=.04). No socio-demographic, clinical, or gallstone characteristics were consistently associated with acute cholecystitis. <h3>Conclusions:</h3> Patients with at least 1 gallstone smaller than 5 mm in diameter have a more than 4-fold increased risk of presenting with acute biliary pancreatitis. A policy of watchful waiting in such cases is unwarranted. Arch Intern Med. 1997;157:1674-1678

This chapter reviews eight years of mammalian cell studies performed by the radiobiology group of Atomic Energy of Canada Ltd., Pinawa, with emphasis on the time scale of radiation-induced events. It reviews this information with other recent studies relating to radiation mechanisms and their time scale. The chapter discusses several concepts currently isolated in separate publications and presents a framework for understanding mechanisms of radiation events in mammalian cells. The chapter discusses the time scale of critical events that may provide a more appropriate conceptual framework for teaching radiation biology, especially to graduate students, than does the usual historical approach. Furthermore, the chapter describes the performance of experiments designed to test some of the mechanisms proposed. It also discusses some implications of these mechanisms for radiation oncology and for the assessment of acceptable radiation exposure limits. It further describes some studies that attempt to define some major pathways that result in radiation-induced loss of cellular proliferative capacity. These are proposed as first approximations to the mechanisms that contribute to proliferative cell death and perhaps to other endpoints.

SummaryThe effectiveness of p-nitroacetophenone (PNAP) as a sensitizing agent to x-rays has been measured in asynchronous and synchronous populations of Chinese hamster cells. PNAP was found to sensitize hypoxic cells selectively (those equilibrated with an environment of 96 per cent N2 + 4 per cent CO2 containing less than 2 p.p.m. O2) with a maximum dose-modifying factor (DMF) of ∼ 1·50 at concentrations of 400 µM and greater in complete medium. In a parallel radiation scheme, the DMF for cells saturated with air was consistently between 2·75 and 3·05. In synchronously-growing Chinese hamster cells, the cyclic variation in surviving fraction measured for cells in air experiencing a radiation dose of 1055 rads could be reproduced for cells irradiated in hypoxic conditions with 2840 rads. We conclude that O2 sensitizes Chinese hamster cells in a dose-modifying fashion, irrespective of position in the cell-cycle. Hypoxic synchronized cells irradiated in the presence of PNAP with 1980 rads are also sensitized in all phases of the cell-cycle, PNAP being slightly more effective for S-phase cells. This differential sensitization is expressed by a two-fold reduction of the cyclic variation in cell sensitivity compared with that of cells irradiated in air or N2.The sensitizing ability of 500 µM PNAP was also determined for air-saturated and hypoxic mouse L-cells, and a selective hypoxic sensitization of ∼ 40 per cent of that of oxygen was measured.

*Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PA; Departments of Radiation Oncology and **Radiological Sciences, Washington University School of Medicine, St. Louis, MO; Imaging Research Laboratory, University of Washington Hospital, Seattle, WA; Department of Nuclear Medicine, Munich Technical University, Munich, Germany; Department of Nuclear Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY; Department of Radiation Oncology, University of Rochester Medical Center, Rochester, NY; Department of Radiation Oncology, Toronto-Sunnybrook Regional Cancer Centre, Toronto, Ontario, Canada

The metabolism-induced binding of 14C-labelled misonidazole (MISO) to hypoxic V79 cells in multicell spheroids has been quantitated using autoradiography. Hypoxia was shown to be the major determinant of the rate of binding. Maximally hypoxic cells bound MISO several times more rapidly than necrotic material in the centre of the spheroids, and up to 50 times more rapidly than well oxygenated cells. The rate of binding to chronically hypoxic cells at the edge of the necrotic centre was 20 times less than to similar cells in other spheroids made maximally hypoxic with N2. This difference is consistent with the greater radio-sensitivity of the chronically hypoxic cells, which is a consequence of their intermediate level of oxygenation. The results indicated that the ability to bind MISO might have considerable potential as a marker for hypoxic cells in tumours. However, some binding patterns cannot be explained by the simplest model of O2 diffusion. It may be necessary to invoke more complex models of O2 diffusion or metabolic gradients within the spheroid which affect the rate of binding.

Misonidazole, as an electron-affinic drug, is capable of accepting electrons from ascorbate, NADPH and a number of enzymes in anoxiic reaction mixtures such as cellular homogenates and microsomal fractions.Although the intracellular location of many of the above electron donors has been established, (usually in microsomes and mitochondria), the sites of covalent binding of metabolically activated misonidazole and its catabolites to cellular macromolecules remain to be elucidated.The biological effects of misonidazole differ quantitatively and possibly qualitatively when cells are incubated in air vis-a-vis hypoxic conditions.Differences in uptake of labeled misonidazole by h!ypoxic or aerobic cells may render this radiosensitizing agent of value in the clinical management of certain tumors.We are currently attempting to establish the intracellular sites of covalent binding of radioactively labeled misonidazole and its catabolites in EMT-6 tumor cells.Single cell suspensions of EMT-6 tumor cells labeled with "C-Misonidazole in hypoxic conditions were homogenized to yield nuclear and cytoplasmic fractions, which were assayed for acid-precipitable radioactivity.The distribution of label bound to various intracellular molecular classes, in both the acid soluble and macromolecular fractions, was determined for EMT-6 cells labeled in air and hypoxia."C-Misonidazole,

Abstract Purpose: 2-Nitro-α-[(2,2,2-trifluoroethoxy)methyl]-imidazole-1-ethanol (TF-MISO) was investigated as a potential noninvasive marker of tissue oxygen levels in tumors using 19F magnetic resonance spectroscopy (MRS) and 19F chemical shift imaging. Experimental Designs: In vitro data were obtained using high-performance liquid chromatography on tumor cells incubated under varying oxygen conditions to determine the oxygen-binding characteristics. In vivo data were obtained using a well-characterized hypoxic murine breast tumor (MCa), in addition to studies on a rat prostate tumor model (R3327-AT) implanted in nude mice. Detection of intratumor 19F signal from TF-MISO was done using MRS for up to 10 h following a 75 mg/kg i.v. injection. Localized distribution of the compound in the implanted MCa tumor has been imaged using slice-selective two-dimensional chemical shift imaging 6 h after injection. Results: The in vitro results showed that TF-MISO preferentially accumulates in cells incubated under anoxic conditions. The in vivo 19F MR spectral features (line width and chemical shift) were recorded as a function of time after injection, and the results indicate that the fluorine atoms are indeed sensitive to changes in the local environment while still providing a detectable MR signal. Ex vivo spectra were collected and established the visibility of the 19F signal under conditions of maximum hypoxia. Late time point (&amp;gt;6 h) tumor tissue concentrations, as obtained from 19F MRS, suggest that TF-MISO is reduced and retained in hypoxic tumor. The feasibility of obtaining TF-MISO tumor distribution maps in a reasonable time frame was established. Conclusions: Based on the results presented herein, it is suggested that TF-MISO has the potential to be a valid magnetic resonance hypoxia imaging reporter for both preclinical hypoxia studies and hypoxia-directed clinical therapy.

The human tolerance to multiple dosages of misonidazole (Ro-07-0582) was studied in 28 patients with different types of malignant neoplasias. The mean total dose for this group of patients was 16.2 g. The main toxicity was peripheral neuropathy with an overall incidence of 35%. This neuropathy occurred more frequently and with greater severity when the drug was administered 3 times a week and when patients received total doses of over 18 g. The best tolerated schedule appears to be once or twice a week up to total dosages of 18 g or less (approximately 11 g/m2). Electron microscopy of a sural nerve biopsy from an affected patient revealed residual of previous distal axonal degeneration, with some segmental demyelination and remyelination, which affected both large and small myelinated nerve fibres.

To test the hypothesis that increasing levels of hypoxia are associated with increased expression of vascular endothelial growth factor (VEGF) in prostate cancer by correlating the level of median tissue oxygenation in human prostate tumors with the immunohistochemically determined level of VEGF expression.Custom-made Eppendorf oxygen microelectrodes were used to quantitate the pO(2) levels in prostate tumors of 13 men undergoing radical prostatectomy. All pO(2) measurements were performed under fluorine-based general anesthesia. Paraffin-embedded tumor tissue from these men was analyzed to measure the level of VEGF expression by immunohistochemical staining. The significance of the associations between the pO(2) levels and VEGF staining were determined by the Pearson correlations.The range of the median pO(2) levels (based on between 97 and 129 individual measurements) among 13 prostate tumors was 0.5 to 44.9 mm Hg. The blinded comparison of pO(2) levels and VEGF staining intensity demonstrated a significant correlation between increasing hypoxia and the percentage of cells staining positive for VEGF (r = -0.721, P = 0.005). This correlation was also significant when pO(2) levels were compared with the overall immunoreactive score, which takes into account staining intensity (r = -0.642, P = 0.018).To our knowledge, this is the first study demonstrating a significant association between increasing levels of hypoxia and increased expression of the angiogenesis marker VEGF in human prostate carcinoma. The results of our study further support the exploration of antiangiogenesis strategies for the treatment of human prostate cancer.

Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential.Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy.OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms.These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

The oxygenation status of tumors may be important for predicting tumor response to therapy. Previous studies with the anaplastic (R3327-AT) and well-differentiated (R3327-H) Dunning rat prostate tumors using indirect assays of tumor oxygenation indicated the relative hypoxic and radioresistant nature of the anaplastic tumor. We now report direct measurements of oxygen in these tumors made with the pO2 histograph to determine: (a) whether a significant difference in oxygenation status could be detected between them: (b) whether sequential measurements on the same tumor gave similar values; and (c) whether tumor oxygenation correlated with tumor volume.R3327-AT and R3327-H tumors were grown in Fischer X Copenhagen rat to volumes of 1.0-7.0 cm3. Electrode measurements (100-200) were made in tumors in anesthetized animals along two parallel tracks. Repeat measurements were made at 1-5 days along different parallel tracks. Oxygen partial pressures of muscle tissue were measured and served as a normal tissue control. Statistical analyses were applied to determine whether tumor oxygen levels were different between the two tumor histologies, whether sequential measurements in the same tumor were reproducible, and whether tumor oxygenation correlated with tumor volume.The average median pO2 of the well-differentiated (n = 15) and the anaplastic (n = 15) tumors was 6.0 mmHg (SE +/- 1.3) and 2.2 mmHg (SE +/- 0.3), respectively. The average median pO2 of normal rat muscle (n = 15) was 23.6 mmHg (SE +/- 2.0). These values represent highly significant differences in oxygen concentration between the two tumors and rat muscle. The differences in average mean pO2 values were also highly significant. Repeat measurements in the same tumors on different days gave average median values of 4.7 and 2.2 mmHg in the R3327-H (n = 15) and R3327-AT (n = 15) tumors, respectively. For these repeat measurements, median pO2 values decreased in 15 and increased in 15 tumors, and were not significantly different from the first measurements. The average differences observed in median pO2 were 37% (SE +/- 7) and 58% (SE +/- 10) for the R3327-H and R3327-AT tumors, respectively. No significant correlation was observed between pO2 levels and the tumor volumes investigated in this study.The median pO2 values of the anaplastic Dunning tumors were significantly lower than those of the well-differentiated tumors (p < 0.001). Oxygen levels in both tumors were significantly lower than those measured in normal rat muscle (p < 0.00005). Repeat measurements of median pO2 in the same tumors were not significantly different for either tumor model (p > 0.5). The changes observed in pO2 distributions within individual tumors from day to day may indicate true dynamics of its oxygenation status and/or the limits of electrode measurements, by sampling along only two insertion sites. The electrode measurements of pO2 in these tumor models are reproducible and confirm previously detected oxygenation differences between the anaplastic and well-differentiated tumors.

The intrinsic radiosensitivity of tumor cells is most frequently reported for asynchronous populations, although cell cycle variation in radiosensitivity is known to be significant. Linear-quadratic analyses of survival data for asynchronous human tumor cells show wide variations in the alpha coefficient with smaller variations in the beta coefficient. HT-29 (colon), OVCAR10 (ovary) and A2780 (ovary) tumor cells with alpha coefficients of 0.03, 0.16 and 0.47 Gy(-1), respectively, and square-root of beta coefficients of 0.23-0.27 Gy(-1) for asynchronous populations were amenable to synchronization by mitotic selection. Selection procedures were optimized for each cell line and produced mitotic populations of >90%, approximately 80% and approximately 65% purity for HT-29, OVCAR10 and A2780 cells, respectively. Mitotic cells from each line exhibited similar and maximum radiosensitivities with alpha coefficients of approximately 1.3 Gy(-1) after irradiation with 137Cs gamma rays and after correction for genome multiplicity. Their relative radiosensitivities observed with asynchronous cells were maintained as they progressed through interphase of the cell cycle. All cells in early G1 phase exhibited a marked radioresistance relative to their sensitivity in mitosis, and maximum interphase radiosensitivity was observed near the G1/S-phase boundary. All cells became increasingly radioresistant as they moved through S phase, the effect being most pronounced for OVCAR10 cells and least pronounced for A2780 cells. HT-29 cells remained relatively radioresistant in G2 phase. The different interphase radiosensitivities observed for these cell lines were determined mainly by the single-hit inactivation mechanism. These studies clearly demonstrate the dominant role of single-hit inactivation in determining the intrinsic radiosensitivity of human tumor cells to 137Cs gamma rays, especially at doses of 2 Gy and less.

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated.Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay.Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells.The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Radiotherapy prescription can now be customized to target the major mechanism(s) of resistance of individual tumors. In that regard, functional imaging techniques should be exploited to identify the dominant mechanism(s). Tumor biology research has identified several mechanisms of tumor resistance that may be unique to radiation treatments. These fall into 3 broad areas associated with (1) tumor hypoxic fraction, (2) tumor growth rate, (3) and the intrinsic radiosensitivity of tumor clonogens. Imaging research has markers in various stages of development for quantifying relevant information about each of these mechanisms, and those that measure tumor oxygenation and predict for radioresistance are the most advanced. Positron-emission tomography (PET) measurement of oxygen 15 has yielded important information, particularly about brain tissue perfusion, metabolism, and function. Indirect markers of tumor hypoxia have exploited the covalent binding of bioreductive intermediates of azomycin-containing compounds whose uptakes are inversely proportional to intracellular oxygen concentrations. Pilot clinical studies with single-photon emission computed tomography (SPECT) and PET detection of radiolabeled markers to tumor hypoxia have been reported. Recently, other studies have attempted to exploit the reduction properties of both technetium and copper chelates for the selective deposition of radioactive metals in hypoxic tissues. A growing number of potentially useful isotopes are now available for labeling several novel chemicals that could have the appropriate specificity and sensitivity. Preclinical studies with "microSPECT" and "microPET" will be important to define the optimal radiodiagnostic(s) for measuring tissue oxygenation and for determining the time after their administration for optimal hypoxic signal acquisition. Radiolabeled markers of growth kinetics and intrinsic radiosensitivity of cells in solid tumors are also being developed. We conclude that radiation oncology is uniquely positioned to benefit from functional imaging markers that identify important mechanisms of tumor radioresistance, since several strategies for overcoming these individual mechanisms have already been identified.

Megakaryocyte (MK) colony formation has been studied in the chronic phase and in the blast crisis of chronic myeloid leukaemia (CML). Blood cells were grown in plasma clot for 13 d. MKs were subsequently identified by immunofluorescent techniques using two monoclonal antiplatelet antibodies (AN51 and J15). The maturation process was studied by ultrastructural methods. A marked increase in the number of circulating CFU‐MK was observed in all the 10 cases studied prior to chemotherapy (70‐fold increase per ml of blood). No significant modification in the regulation of MK colony formation as compared to that of normal subjects was observed. The predominant abnormality in maturation in culture was the occurrence of many hypoploid MKs (microMKs). However, the cytoplasmic maturation of the MKs was identical to that of normal subjects with occasional platelet shedding. Since microMKs predominated in some patients, scoring of MK colonies in CML necessitated immunofluorescent labelling to permit identification of MKs. During the blast crisis, MK colony formation occurred in four out of five patients with an extremely high plating efficiency in the case of promegakaryoblastic transformation. In contrast, MK colonies could not be grown from blood samples of patients with acute leukaemia, including two cases of promegakaryoblastic leukaemia. Maturation of MKs in blast crisis was identical to that of the chronic phase. Furthermore, after short periods of culture in liquid medium, circulating promegakaryoblasts from spontaneously to become large MKs exhibiting demarcation membranes and α‐granules, while those from two cases of megakaryoblastic leukaemias did not mature. In consequence, the blast crisis of CML exhibits a different culture pattern from acute leukaemia. These results suggest, therefore, that the acute transformation of CML cannot be simply explained by clonal changes and that environmental and regulatory factors could be also involved.

Dunning R3327-H and R3327-AT tumors growing subcutaneously in the flanks of Fischer X Copenhagen rats were irradiated with 137Cs gamma-rays at volumes of approximately 300 mm. The effects of various doses of radiation were estimated by measurements of subsequent tumor growth as well as by histological evaluation. The well-differentiated, hormonally-responsive R3327-H tumor was more radiosensitive than the anaplastic R3327-AT tumor. The reasons for this increased radiation sensitivity of the R3327-H tumor include a greater "apparent" radiation sensitivity of tumor cells and the absence of tumor hypoxia. The presence of hypoxic tumor stem cells was inferred from significant radiosensitization of tumor growth delay by 0.5 mg./gm. misonidazole and by a technique which utilizes radioactively-labelled misonidazole as a marker for hypoxic cells. The persistence of a mass of R3327-H tumor tissue after aggressive radiotherapy was not indicative of tumor cells. Furthermore, the rapid increase in volume of R3327-AT cells after aggressive radiotherapy was attributed to limited proliferation of tumor cells which were destined to ultimately die. Possible implications of these findings for the management of human prostatic adenocarcinoma are discussed.

When a heart is in a stable inotropic state, the end-systolic pressure-volume points of each work cycle fall on a straight line regardless of the magnitude of the afterload or the initial end-diastolic volume: cardiac O2 consumption (MVO2) per beat is linearly correlated with ventricular systolic pressure-volume area (PVA), defined in terms of stroke work and potential energy components. Moreover, if the basal and activation components of the cardiac energy cycle are subtracted, hearts operate at a constant PVA/MVO2 efficiency. The present review examines the energetic implications of these results for current muscle models, discussing the energetic background of earlier skeletal muscle viscoelastic models and examining differences between the vectorial outputs of ion transport ATPases and myofibrillar ATPases. The PVA data point to a unique stoichiometric relationship between myocardial energy flux and vectorial output, and it is shown that most existing myocardial O2 consumption data can be reconciled with the PVA concept. However, most muscle models would not predict a linear stoichiometric relation between energy flux and pressure-volume potential energy. We pose the question as to whether there is an undiscovered autoregulatory process at work in muscle.

Activation of reovirus transcriptase activity, latent in intact virions, by digestion of purified virions with chymotrypsin (CHT) in vitro shows a stringent requirement for specific monovalent cations. Cs + , Rb + , or K + ions are capable of facilitating activation by chymotryptic digestion. Na + , Li + , or NH 4 + ions are not capable of facilitating the CHT activation of polymerase activity and are antagonistic towards the effects of the facilitating ions. The data indicate that the effect of the cations is exerted on activation of the polymerase activity by CHT as opposed to an effect on polymerization per se. This effect may be important biologically in that it provides a mechanism whereby the virion can sense whether it is in an intracellular or an extracellular environment and thereby can avoid premature uncoating.

The optical properties of tumor tissue provide important information for optimizing treatment plans in photodynamic therapy, especially when interstitial application by multiple fibers is planned. Near infrared light, required to activate novel photosensitizers, should facilitate improved light penetrance of tumor tissue compared with 630 nm light used for activating Photofrin II. We have measured light energy fluence rates for 630 and 789 nm light along radial tracks from a single laterally diffusing optical fiber centrally implanted into Dunning R3327-AT and R3327-H rat prostate tumors in anesthetized rats. A total of 20 R3327-AT and 10 R3327-H tumors were used in this study with volumes from 2.6 to 13.3 cm3. Light track data were analyzed by an empirical model that described light attenuation. At 630 nm, light attenuation coefficients (LAC) were approximately 1.9 x higher than those at 789 nm for both tumors with the well-differentiated, well-perfused tumor (R3327-H) attenuating to a greater extent than did the rapidly growing anaplastic tumor (R3327-AT). The intertumor variation of LAC was greater than the spatial variations observed within individual tumors. LAC were a function of tumor volume for only 630 nm light in the R3327-AT tumors.

The surviving fraction of human tumor cell lines after 2 Gy (SF2) varies between 0.1 and 0.8. It has been postulated that differences in inherent radiosensitivity of tumor cells are a major determinant of radiation response in vivo. Assays of inherent radiosensitivity based on acute survival are being developed as predictors of tumor response which often assume that the same inherent radiosensitivity persists throughout a fractionated treatment. We have investigated the response of 2 human tumor cell lines (A549 and MCF7) with different inherent radiosensitivities to in vitro fractionated irradiation. A549 cells had an SF2 of 0.62 and a mean inactivation dose (D) of 3.07 Gy whereas MCF7 cells had an SF2 of 0.30 and a D of 1.52 Gy. Split dose repair capacity (at equal survival levels) was less for A549 than for MCF7 cells and recovery kinetics for both cell lines were substantially longer than those of rodent cell lines. Survival after 5 fractions of 2 Gy given 12 hr apart at 37 degrees C was near to that predicted from the acute survival curve, assuming complete repair and no proliferation. Acute survival of A549 cells which survived 5 fractions of 2 Gy given 12 hr apart was similar to the acute survival of unirradiated cells. When A549 cells were incubated at 22 degrees C between 5 fractions of 2 Gy given 12 hr apart, proliferation and split dose repair were substantially inhibited. These studies support the proposals to use in vitro inherent radiosensitivity assays for the prediction of in vivo response of tumors to fractionated treatment.

Misonidazole (MISO), a hypoxic cell radiosensitizer, forms covalently-linked adducts to cellular molecules as a result of bioreductive metabolism, a process which is strongly dependent upon oxygen concentration. MISO binding to liver tissue taken from air-breathing mice was three to five times greater than binding to other normal tissues. The relative binding of [14C]MISO to various mouse tissue cubes in vitro was measured by autoradiography as a function of defined oxygen concentrations, and standard curves (binding rate vs oxygen concentration) were generated. The oxygen concentration for half-maximum binding as well as the maximum and minimum binding rates (grains per 100 μm2) observed for liver tissue were not significantly different from those measured for brain or heart tissue. These results, along with previously published data on MISO binding to isolated hepatocytes in vitro, suggest that the elevated binding to liver in vivo may result, in part, from the organ existing at a significantly lower pO2 than other normal tissues. They also suggest that this drug adduct procedure could be developed as a sensitive method for the quantitative measurement of tissue pO2 at the cellular level.

Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and ...Read More

Journal Article The effect of metabolic substrate on mechanical activity and heat production in papillary muscle Get access J. BRIAN CHAPMAN, J. BRIAN CHAPMAN From the Department of Physiology, Monash University, Clayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar COLIN L. GIBBS COLIN L. GIBBS From the Department of Physiology, Monash University, Clayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Cardiovascular Research, Volume 8, Issue 5, September 1974, Pages 656–667, https://doi.org/10.1093/cvr/8.5.656 Published: 01 September 1974

Tumour oxygenation status in individual patients may be assessed using the bioreduction and linkage of 2-nitroimidazole markers to viable hypoxic cells in vivo with subsequent detection by conventional nuclear medicine techniques. Iodoazomycin arabinoside (IAZA) was radiolabelled with Iodine-123 and administered i.v. to 51 patients with newly diagnosed malignancies whose tumours were subsequently imaged by planar and single-photon emission computed tomographic (SPECT) procedures. Quantitative analyses of radiotracer avidity were performed at 24 h post-injection and tumour-normal tissue ratios of greater than 1.10 were deemed positive for tumour hypoxia. By this criterion, the frequencies of hypoxia in small-cell lung cancer, squamous cell carcinomas of head and neck and malignant gliomas were 60% (9/15), 40% (6/15) and 0% (0/11) respectively. The correlation of positive IAZA scans with tumour control and survival in patients with lung cancer and head and neck tumours is currently under study. Preliminary observations in neck metastases from squamous cell carcinoma of head and neck tumours indicates decreased local control at 3 months post-treatment in tumours with IAZA avidity. This study concludes that: (1) 123I-IAZA can be administered safely and repeatedly as an outpatient routine imaging procedure in cancer patients during initial work-up and follow-up; (2) that retained drug can be detected by conventional nuclear medicine procedures in inaccessible deep-seated tumours; and (3) that this technique could prove useful for identifying those patients for whom hypoxia-directed therapy is indicated.

CancerVolume 89, Issue 9 p. 2018-2024 Original ArticleFree Access Increasing levels of hypoxia in prostate carcinoma correlate significantly with increasing clinical stage and patient age An Eppendorf pO2 study B. Movsas M.D., Corresponding Author B. Movsas M.D. b_movsas@fccc.edu Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania Fax: (215) 214-1629Department of Radiation Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111===Search for more papers by this authorJ. D. Chapman Ph.D., J. D. Chapman Ph.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorR. E. Greenberg M.D., R. E. Greenberg M.D. Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorA. L. Hanlon Ph.D., A. L. Hanlon Ph.D. Department of Radiation Oncology (Statistics), Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorE. M. Horwitz M.D., E. M. Horwitz M.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorW. H. Pinover D.O., W. H. Pinover D.O. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorC. Stobbe M.S., C. Stobbe M.S. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorG. E. Hanks M.D., G. E. Hanks M.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this author B. Movsas M.D., Corresponding Author B. Movsas M.D. b_movsas@fccc.edu Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania Fax: (215) 214-1629Department of Radiation Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111===Search for more papers by this authorJ. D. Chapman Ph.D., J. D. Chapman Ph.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorR. E. Greenberg M.D., R. E. Greenberg M.D. Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorA. L. Hanlon Ph.D., A. L. Hanlon Ph.D. Department of Radiation Oncology (Statistics), Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorE. M. Horwitz M.D., E. M. Horwitz M.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorW. H. Pinover D.O., W. H. Pinover D.O. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorC. Stobbe M.S., C. Stobbe M.S. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this authorG. E. Hanks M.D., G. E. Hanks M.D. Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PennsylvaniaSearch for more papers by this author First published: 30 October 2000 https://doi.org/10.1002/1097-0142(20001101)89:9<2018::AID-CNCR19>3.0.CO;2-YCitations: 26 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract BACKGROUND The purpose of this study was to analyze the extent of hypoxia in prostate carcinoma tumors using the Eppendorf pO2 microelectrode and correlate this with pretreatment characteristics and prognostic factors. METHODS Custom-made Eppendorf pO2 microelectrodes were used to obtain pO2 measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies) as well as from a region of normal muscle for comparison. Each set of measurements comprised approximately 100 separate readings of pO2, for a total of 10,804 individual measurements. Fifty-five patients with localized prostate carcinoma were studied: Forty-one patients received brachytherapy implants, and 14 patients underwent radical prostatectomy. The pO2 measurements were obtained in the operating room by using a sterile technique under spinal anesthesia for the brachytherapy group and under general anesthesia for the surgery group. The Eppendorf histograms were recorded and described by the median pO2, mean pO2, and percentage < 5 mm Hg and < 10 mm Hg. A multivariate mixed-effects analysis for the prediction of tumor oxygenation was performed and included the following covariates: type of tissue (prostate vs. muscle), type of treatment (implant vs. surgery) and/or anesthesia (spinal vs. general), prostate specific antigen level, disease stage, patient age and race, tumor grade, tumor volume, perineural invasion, and hormonal therapy. RESULTS Due to differences in patient characteristics and the anesthesia employed, control measurements were obtained from normal muscle (in all but two patients). This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median pO2, 9.9 mm Hg) compared with the measurements normal muscle (average median pO2, 28.6 mm Hg; P < 0.0001). A multivariate, linear, mixed analysis demonstrated that, among all of the patients, the significant predictors of oxygenation were tissue (prostate vs. muscle) and anesthesia (spinal vs. general) or treatment (implant vs. surgery). Among the brachytherapy (spinal anesthesia) patients, the significant predictors of pO2 were tissue type, disease stage, and patient age. There were no significant predictors of oxygenation in the surgical (general anesthesia) group. CONCLUSIONS This study, employing in vivo electrode oxygen measurements, demonstrated that hypoxia exists in prostate carcinoma tumors. A dramatic effect of anesthesia was observed, likely due to modulation of polarography in the presence of fluorine. Within the group of brachytherapy (spinal anesthesia) patients, increasing levels of hypoxia (within prostatic tissue) correlated significantly with increasing clinical stage and patient age. More patients will be accrued to this prospective study to further correlate the oxygenation status in prostate carcinoma tumors with known prognostic factors and, ultimately, treatment outcome. Cancer 2000;89:2018–24. © 2000 American Cancer Society. REFERENCES 1 Movsas B, Hanlon A, Teshima T, Hanks GE. Analyzing predictive models following definitive radiotherapy for prostate carcinoma. Cancer 1997; 80: 1093– 102. 2 Gatenby R, Kessler H, Rosenblum J, Coia LR, Moldofsky PJ, Hartz WH, et al. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys 1988; 14: 831– 8. 3 Hockel M, Knoop C, Schlenger K, Vondran B, Baussman E, Mitze M, et al. Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix. 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Conformal high dose rate Ir-192 boost brachytherapy in locally advanced prostate cancer: superior prostate specific antigen response compared to external beam treatment. Cancer J Sci Am 1997; 3: 346– 52. 19 Herold DM, Hanlon AL, Movsas B, Hanks GE. Age-related prostate cancer metastases. Urology 1998; 51(6): 985– 99. 20 Carter HB, Epstein JI, Partin AW. Influence of age and prostate-specific antigen on the chance of curable prostate cancer among men with nonpalpable disease. Urology 1999; 53: 126– 30. 21 Ripple M, Henry W, Rago R, Wilding G. Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells. J Natl Cancer Inst 1997; 89(1): 40– 8. 22 Joseph I, Isaacs J. Potentiation of the antiangiogenic ability of linomide by androgen ablation involves down-regulation of vascular endothelial growth factor in human androgen-responsive prostatic cancers. Cancer Res 1997; 57(6): 1054– 7. 23 Yeh KA, Biade S, Lanciano M, Brown DQ, Fenning MC, Babb JS, et al. Polarographic needle electrode measurements of oxygen in rate prostate carcinomas; accuracy and reproducibility. Int J Radiat Oncol Biol Phys 1995; 33(1): 111– 8. 24 Vaupel P, Schlenger K, Knoop C, Hockel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 1991; 51: 3316– 22. 25 Cruickshank G, Rampling R, Cowan W. The pO2 distribution of human brain tumors determined by fine needle polarography. Adv Exp Med Biol 1994; 345: 465– 70. 26 Teicher B, Schwartz G, Dupuis N, et al. Oxygenation of human tumor xenografts in nude mice by a perfluorochemical emulsion and carbogen breathing. Artificial Cells Blood Subst Immobil Biotechnol 1995; 22: 1369– 75. 27 Groshar D, McEwan A, Parliament M, Urtasan RC, Golberg LE, Hoskinson M, et al. Imaging tumor hypoxia and tumor perfusion. J Nucl Med 1993; 34: 885– 8. 28 Urtasun R, McEwan A, Parliament M, et al. Measurement of hypoxia in human tumors by SPECT imaging of iodoazomycin arabinoside. Br J Cancer 1996; 74: 209– 12. 29 Rasey J, Koh W, Evans M, et al. Quantifying regional hypoxia in human tumors with positron emission tomography of 18F fluoromisonidazole: a pretherapy study of 37 patients. Int J Radiat Oncol Biol Phys 36: 417– 28. 30 Chapman J, Engelhardt E, Stobbe C, Schneider RF, Hanks GE. Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays. Radiother Oncol 1998; 46: 229– 37. Citing Literature Volume89, Issue91 November 2000Pages 2018-2024 ReferencesRelatedInformation

AbstractPurpose : To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day −1 fractions and to low dose-rate brachytherapy. Materials and methods : Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the α- and β-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of β-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. Results : The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the β-mechanism. α-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high α-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Conclusions : Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (α) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of β-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

Nitroimidazole drugs were initially developed as selective radiosensitizers of hypoxic cells and, consequently, as adjuvants to improve the local control probabilities of current radiotherapies. Misonidazole (MISO), the prototype radiosensitizing drug, was found in Phase I clinical studies to cause dose-limiting neurotoxicities (mainly peripheral neuropathies). MISO was also found to be cytotoxic in the absence of radiation and to covalently bind to cellular molecules, both processes demonstrating rates much higher in hypoxic compared with oxygenated cells. It is likely that neurotoxicity, cellular cytotoxicity and adduct formation results from reactions between reduction intermediates of MISO and cellular target molecules. Spin-offs from radiosensitizer research include the synthesis and characterization of more potent hypoxic cytotoxins and the exploitation of sensitizer-adducts as probes for measuring cellular and tissue oxygen levels. Current developments in hypoxic cell cytotoxin and hypoxic cell marker research are reviewed with specific examples from studies which characterize the cellular reduction of TF-MISO, (1-(2-nitro-l-imidazolyl)-3[2,2,2-trifluoroethoxy]-2-propanol).

Growth of megakaryocyte colonies from human bone marrow progenitors has been achieved in plasma clot culture. Megakaryocyte colonies were identified either by cytological examination or by immunofluorescent labelling using a monoclonal antiplatelet antibody (J 15). No significant differences were observed in the quantitation of the colonies by these two methods. In the absence of a stimulating factor, MK colonies were detectable when high cellular concentrations were seeded. Among the numerous conditioned media tested for their ability to stimulate MK colony formation, conditioned medium from leukocytes stimulated by PHA (PHA-LCM) was the most effective. However, under standard conditions of culture corresponding to 20% normal human sera, colony formation was not related linearly to seeding density even when cultures were stimulated by PHA-LCM. Upon reduction of the concentration of serum (2.5-5%) in the culture medium, colony formation displayed a linear relationship seeding density only when PHA-LCM was used as the stimulating factor. At the same time, the size of the colonies increased. Such inhibition was observed with all the human sera tested but it varied in extent from one batch to another. Replacement of serum by albumin, iron-saturated transferrin, alpha-thioglycerol and low density lipoproteins at physiological concentration but in the presence of bovine plasma provided a culture system whose ability to support colony formation equalled that of low concentrations of whole serum; spontaneous MK colony formation still occurred. Our results provide evidence for the presence of an inhibitor(s) of MK colony formation in normal human sera, and demonstrate the role of cellular factors in stimulating MK colony formation.

ARTICLESEffects of stimulus conditions, temperature, and length on energy output of frog and toad sartoriusCL Gibbs, and JB ChapmanCL Gibbs, and JB ChapmanPublished Online:01 Oct 1974https://doi.org/10.1152/ajplegacy.1974.227.4.964MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByEfficiency of Skeletal MuscleThe basis of differences in thermodynamic efficiency among skeletal muscles16 October 2017 | Clinical and Experimental Pharmacology and Physiology, Vol. 44, No. 12Review on biothermoydnamics applications: timeline, challenges, and opportunities26 January 2017 | International Journal of Energy Research, Vol. 41, No. 11Effects of temperature and force requirements on muscle work and power outputJournal of Experimental Biology, Vol. 220, No. 11Thermodynamic analysis of the squid mantle muscles and giant axon during slow swimming and jet escape propulsionEnergy, Vol. 102Conquering the world in leaps and bounds: hopping locomotion in toads is actually bounding28 March 2015 | Functional Ecology, Vol. 29, No. 10A review of the thermal sensitivity of the mechanics of vertebrate skeletal muscle13 March 2013 | Journal of Comparative Physiology B, Vol. 183, No. 6Is the efficiency of mammalian (mouse) skeletal muscle temperature dependent?30 September 2010 | The Journal of Physiology, Vol. 588, No. 19The scaling of myofibrillar actomyosin ATPase activity in apid bee flight muscle in relation to hovering flight energetics12 March 2010 | Journal of Experimental Biology, Vol. 213, No. 7Effects of the transition time between muscle-tendon stretch and shortening on mechanical efficiency17 January 2006 | European Journal of Applied Physiology, Vol. 96, No. 6The efficiency of muscle contractionProgress in Biophysics and Molecular Biology, Vol. 88, No. 1Cardiac energetics: Sense and nonsenseClinical and Experimental Pharmacology and Physiology, Vol. 30, No. 8In situ rat fast skeletal muscle is more efficient at submaximal than at maximal activation levelsF. Abbate, C. J. De Ruiter, C. Offringa, A. J. Sargeant, and A. De Haan1 May 2002 | Journal of Applied Physiology, Vol. 92, No. 5Energetics of crossbridge phosphorylation and contraction in vascular smooth muscle.Hypertension, Vol. 23, No. 6_pt_2Transducing Chemical Energy into Mechanical Function: A Comparative View14 January 2012Physiological features of the opercularis muscle and their effects on vibration sensitivity in the bullfrog Rana catesbeiana1 September 1987 | Journal of Experimental Biology, Vol. 131, No. 1Thermal dependence of maximum Ca2+-activated force in skinned muscle fibres of the toad Bufo marinus acclimated at different temperaturesJournal of Experimental Biology, Vol. 129, No. 1Cardiac energetics and the Fenn effectCardiac energeticsA comparison between field habits and contractile performance of frog and toad sartorius muscleJournal of Comparative Physiology ? B, Vol. 151, No. 2The effects of temperature on the energetics of rat papillary musclePfl�gers Archiv European Journal of Physiology, Vol. 379, No. 2Oxygen Consumption and Lactate Production of the Rat Portal Vein in Relation to its Contractile ActivityActa Physiologica Scandinavica, Vol. 100, No. 1 More from this issue > Volume 227Issue 4October 1974Pages 964-971 Copyright & PermissionsCopyright © 1974 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1974.227.4.964PubMed4215327History Published online 1 October 1974 Published in print 1 October 1974 Metrics

Two recent editorials in this Journal about the linear quadratic (LQ) model (1Glatstein E. The omega on alpha and beta.Int J Radiat Oncol Biol Phys. 2011; 81: 319-320Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 2Brenner D.J. Sachs R.K. Peters L.J. et al.We forget at our peril the lessons built into the α/β model.Int J Radiat Oncol Biol Phys. 2012; 82: 1312-1314Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) have debated its use on essentially empirical grounds. Missing are its roots in biophysics and what they state about how it can (and cannot) be used. In the 1970s, 2 roads led to analysis of in vitro cell killing by ionizing radiation in terms of the underlying mechanisms. Kellerer and Rossi (3Kellerer A.M. Rossi H.H. The theory of dual radiation action.Curr Topics Radiat Res Q. 1972; 8: 85-158Google Scholar) explored the statistics of energy deposition in small volumes—the microdosimetric approach. Chadwick and Leenhouts (4Chadwick K.H. Leenhouts H.P. A molecular theory of cell survival.Phys Med Biol. 1973; 18: 78-87Crossref PubMed Scopus (333) Google Scholar) related cell killing to lesions in 2-stranded DNA—the biophysical approach. Both approaches postulate that radiation can kill cells by 2 distinct processes, a 1-hit mechanism and another that requires 2 independent events. We began to use the LQ equation in 1973 for the pragmatic reason that it fits the survival data better than the other models we tested (5Gillespie C.J. Chapman J.D. Reuvers A.P. et al.The inactivation of Chinese hamster cells by X-rays: synchronized and asynchronous cell populations.Radiat Res. 1975; 65: 353-364Crossref Scopus (73) Google Scholar). It soon emerged that, if key factors were controlled, this equation offers insights into the mechanisms involved in cell death and survival during and after irradiation. Here, we reviewed 4 forms of this equation that describe cell survival in specific circumstances and how they might help those working to improve radiotherapeutic protocols. The basic LQ equation states that the cell surviving fraction (S) is the product of 2 Poisson escape probabilities. The mean numbers of events for the underlying mechanisms are proportional to the first and second powers of dose, D, respectively.S=exp(−αD)×exp(−β0D2)LQ (1) The sublesions associated with β0-inactivation (likely single-strand breaks [6Dugle D.L. Gillespie C.J. Chapman J.D. DNA strand breaks, repair and survival in X-irradiated mammalian cells.PNAS. 1976; 73: 809-812Crossref PubMed Scopus (121) Google Scholar]) repair rapidly at mammalian body temperatures. Thus, to obtain precise values of α and β0, cells are irradiated at dose rates >10 Gy/min or at 2°C-6°C, such that the first sublesion of a 2-hit event will not be removed before a second has been induced. The sources available to most radiobiology research have dose rates of 1-2 Gy/min at positions convenient for cell irradiation; thus, low temperatures should be used to obtain the maximal and true value of β0. This basic LQ equation can be written in a form that yields a straight line such that the 2 parameters are the intercept at 0 dose and the slope, respectively.−ln(S)/D=α+β0DLQ (1a) The basic LQ equation applies to cell populations of sufficiently homogeneous radiosensitivity. Thus, much of our research investigated the cell-survival and cell-killing mechanisms with synchronized cells in mitosis (tetraploid) or in G1 phase (diploid) of the cell cycle. Stationary (G0) phase cells (diploid) were also used in experiments in which it was not feasible to synchronize the cells. In this model, the α/β ratio defines the dose at which the different mechanisms contribute equally to cell killing. However, most radiobiology research uses asynchronous populations of normal or tumor cell lines whose survival can also be characterized with an LQ equation. Although LQ Eq. 1 can be fitted to such data, the resultant α and β parameters do not precisely express the underlying mechanisms. Rather, they are associations of values for subpopulations in which the range of α values affects the fitted β values and vice versa (5Gillespie C.J. Chapman J.D. Reuvers A.P. et al.The inactivation of Chinese hamster cells by X-rays: synchronized and asynchronous cell populations.Radiat Res. 1975; 65: 353-364Crossref Scopus (73) Google Scholar). This is reflected by a different form of the equation:S=exp(−α¯D)×exp(−β0¯D2)=∑xnx[exp(−αxD)×exp(−β0xD2)]LQ (2) where nx is the fraction of cell population x, with parameters αx and β0x, and α¯ and β0¯ are effective values for the mixed population. The mechanistic ambiguity of the parameter values from mixtures of cells of different radiosensitivity should be clear. This form of the equation will be useful for asynchronous cell populations, for mixtures of oxygenated and hypoxic cells, for mixtures of quiescent and proliferating cells, for cells in planning tumor volume voxels exposed to particles of the variable linear energy transfer, and so forth. The variation in α¯ for various human tumor cell lines correlates well with the different radioresponses of the tumors from which they were derived; in contrast, β0¯ varies only slightly among cell types (7Deacon J. Peckham M.J. Steel G.G. The radioresponsiveness of human tumors and the initial slope of the cell survival curve.Radiother Oncol. 1984; 2: 317-323Abstract Full Text PDF PubMed Scopus (416) Google Scholar, 8Chapman J.D. Single-hit mechanism of tumor cell killing by radiation.Int J Radiat Biol. 2003; 79: 71-81PubMed Google Scholar). Cell killing by radiation was also characterized at intermediate dose rates and different temperatures. These studies identified a first-order repair process that accounts for the reduced cell inactivation observed (9Chapman J.D. Gillespie C.J. Radiation induced events and their time scale in mammalian cells.Adv Radiat Biol. 1981; 9: 143-198Crossref Google Scholar). This effect can be expressed in another form of the basic equation:S=exp(−α¯D)×exp(−β0¯D2{1−[1−exp(−m)]/m}/m)LQ (3) where α and β0 are unchanged, m is the ratio kD/R, k is the measured rate constant for repair of sublesions at the specific temperature of interest, and R is dose rate. This form of the equation should be used when low dose rates or prolonged exposures are used. It tends toward a straight line at a high dose on a semi-log plot. Our studies yielded a value of k = 0.03/min at 37°C for Chinese hamster fibroblasts (9Chapman J.D. Gillespie C.J. Radiation induced events and their time scale in mammalian cells.Adv Radiat Biol. 1981; 9: 143-198Crossref Google Scholar); however, human tumor cell lines might have different repair rates. Nevertheless, this form of the equation quantitatively expresses the reduced cell killing due to repair of sublesions during exposure of more than a few minutes, which should come as no surprise. Additional research might indicate that an additional and much slower first-order repair process, such as is observed for single-strand break repair (6Dugle D.L. Gillespie C.J. Chapman J.D. DNA strand breaks, repair and survival in X-irradiated mammalian cells.PNAS. 1976; 73: 809-812Crossref PubMed Scopus (121) Google Scholar), might result in reduced cell killing at low dose rates. Those working to introduce radiobiology parameters into treatment planning protocols need another form of the basic equation. Because most radiation therapy is delivered in daily fractions, 5 d/wk, there is enough time between dose fractions for complete repair of the sublethal damage associated with the 2-hit mechanism and for clonogen reassortment. Thus, fractionated doses will produce cell killing, as described by the following equation:S=exp[−n(α¯d+β0¯d2)]LQ (4) where n fractions of dose d deliver a total dose D = nd. On a plot of log(S) vs D, these survival curves also take on a linear appearance over several decades of cell kill (10Nahum A.E. Movsas B. Horwitz E.M. et al.Incorporating clinical measurements of hypoxia into tumor local control modeling of prostate cancer: implications for the α/β ratio.Int J Radiat Oncol Biol Phys. 2003; 57: 391-401Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). If one knows the total number and the intrinsic radiosensitivity of clonogens in a group of similar tumors, the tumor control probability models can predict local tumor control as a function of the total dose. The radiation inactivation parameters for pathologically similar tumors in different patients or for clonogens in individual tumors are known to vary (11Nahum A.E. Sanchez-Nieto B. Tumor control probability modeling: basic principles and applications in treatment planning.Phys Med. 2001; 17: 369-380Google Scholar). Consequently, guesstimates are made for specific tumor-treatment cases and controversy can ensue. A case in point, the analysis of prostate cancer brachytherapy yielded excellent results when a very low value for α (0.036 Gy−1) and 15 ± 2 clonogens were assumed for tumors with Gleason scores of mainly 6 and 7 (12Brenner D.J. Hall E.J. Fractionation and protraction for radiotherapy of prostate carcinoma.Int J Radiat Oncol Biol Phys. 1999; 43: 1095-1101Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Several pathologists and urologists consider this clonogen number to be low for tumors of this volume. Equally good fits to these clinical data were obtained using average values of α and β for prostate cancer cell lines reported in published studies, with clonogen numbers of 1-10 million and the recently obtained knowledge of severe hypoxia in some human prostate cancers (10Nahum A.E. Movsas B. Horwitz E.M. et al.Incorporating clinical measurements of hypoxia into tumor local control modeling of prostate cancer: implications for the α/β ratio.Int J Radiat Oncol Biol Phys. 2003; 57: 391-401Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). That such widely disparate values can produce equally good fits to clinical data underlines the need for better tumor biology information. Additional studies could fine tune the more important parameters such as α¯ and β¯, with some estimate of errors, quiescent vs. proliferating compartments, oxygenated vs hypoxic cells, sublethal damage repair rates, and clonogen number. With realistic parameters, radiation oncology could go beyond empiricism to tumor control probability modeling to predict confidently the relative merits of fractionation schemes and to better understand the 4 or 5 Rs of radiobiology that apply to tumor radiation therapy (13Withers H.R. The four R's of radiotherapy.Adv Radiat Biol. 1975; 5: 241-271Crossref Google Scholar, 14Steel G.G. Peacock J.H. Why are some human tumors more radiosensitive than others?.Radiother Oncol. 1989; 15: 63-72Abstract Full Text PDF PubMed Scopus (62) Google Scholar). As for normal tissue complication probability modeling, even less is known of the underlying mechanisms of normal tissue damage that account for deleterious effects of radiation. If the damage results from killing stem cells in the tissue, for example, quantitative LQ radiobiology could come into play. However, when the late effects of radiation involve interactions between stem cell, stromal, and vascular elements in the tissues, it is unclear how the LQ analyses might apply. Most current radiobiologic research does not attempt to meet the conditions for LQ analysis and thus does not readily relate to underlying physical mechanisms. Our research has suggested that single-hit inactivation by X-rays results from electron track ends depositing energy in compacted chromatin (with some indication of a lipid/DNA composition), producing nonreparable lesions (15Chapman J.D. Biophysical models of mammalian cell inactivation by radiation.in: Meyn R.E. Withers H.R. Radiation Biology in Cancer Research. Raven Press, New York1980: 21-32Google Scholar). However, 2-hit inactivation results from simple DNA lesions (mostly reparable strand breaks) produced by simple ionizations (∼60 eV), those that radiation chemists term “spurs.” Hydroxyl radicals generated in cellular water mediate a large proportion of the killing of cells by both mechanisms (9Chapman J.D. Gillespie C.J. Radiation induced events and their time scale in mammalian cells.Adv Radiat Biol. 1981; 9: 143-198Crossref Google Scholar). How these different lesions can produce the aberrations observed in the mitotic chromosomes of irradiated cells has been elegantly described (16Chadwick K.H. Leenhouts H.P. The Molecular Theory of Radiation Biology. Springer-Verlag, New York1981Crossref Google Scholar). Using the appropriate LQ equation, this biophysical understanding of tumor cell killing by ionizing radiation can be extremely useful in the design of improved treatment protocols. For example, hypofractionated radiation therapy causes less stress to the patient (shorter overall treatment times) and has lower cost (fewer patient setups) and thus should be studied vigorously. Current treatments with 1.8-2.0 Gy/fraction were optimized empirically using inferior imaging and radiation delivery technologies. Because the 2 cell-killing mechanisms exhibit entirely different properties (8Chapman J.D. Single-hit mechanism of tumor cell killing by radiation.Int J Radiat Biol. 2003; 79: 71-81PubMed Google Scholar), LQ analysis can be used to usefully guide adjustments to current protocols in light of the expected biologic consequences. The previous radiation biophysic studies of tumor cell killing should be embraced and the appropriate LQ equations exploited for the design of improved treatment strategies.

The radiation killing of tumor cells by ionizing radiation is best described by the linear-quadratic (LQ) model.Research into the underlying mechanisms of αand β-inactivation has suggested that different molecular targets (DNA in different forms) and different microdosimetric energy deposits (spurs versus electron trackends) are involved.Clinical protocols with fractionated doses of about 2.0 Gy/day were defined empirically, and we now know that they produce cancer cures mainly by the α-inactivation mechanism.Radiobiology studies indicate that α and β mechanisms exhibit widely different characteristics that should be addressed upfront as clinical fractionation schemes are altered.As radiation treatments attempt to exploit the advantages of larger dose fractions over shorter treatment times, the LQ model can be used to predict iso-effective tumor cell killing and possibly iso-effective normal tissue complications.Linking best estimates of radiobiology and tumor biology parameters with tumor control probability (TCP) and normal tissue complication probability (NTCP) models will enable us to improve and optimize cancer treatment protocols, delivering no more fractions than are strictly necessary for a high therapeutic ratio.

: To determine the relative effectiveness of specific cellular reductases for the activation and binding of 2-nitroimadazoles in vivo. : Monkey kidney cells were transfected with recombinant plasmids to effect intracellular overexpression of P450 reductase and DT-diaphorase. The covalent binding of 2-nitroimidazoles to cellular macromolecules was measured as a function of time of cell incubation at various oxygen concentrations. The effect of allipurinol on cellular binding of radiolabeled 2-nitroimidazoles was also measured. : A 1,000-fold overexpression of DT-diaphorase resulted in a small but significant increase in 2-nitroimidazole binding rate. An 80-fold overexpression of cytochrome P450 reductase resulted in a 5–7-fold increase in the binding rate of 2-nitroimidazole. The inhibition of xanthine oxidase by allipurinol had no effect on 2-nitroimidazole binding rates. The amplification of P450 reductase activity within cells was always much larger than the resultant increase in 2-nitroimidazole binding rate, suggesting an enzyme kinetic process less than first order and possibly of . : These data suggest that cytochrome P450 reductase is the most important enzyme in these cells for reducing 2-nitroimidazoles to intermediates which can covalently bind to cellular macromolecules. Furthermore, since this cellular process demonstrates kinetics, a tissue's capacity for binding 2-nitroimidazole drug in hypoxia should be proportional to the square root of its intracellular P450 reductase level.

Dunning R3327-AT prostate carcinomas growing in Fischer X Copenhagen rats were treated with interstitial photodynamic therapy (PDT--15 mg/kg Photofrin II 4 hours before illumination with 630-nm light via four parallelly implanted optical fibers) at different light intensities. Forty to 60 minutes after treatment, 31P-nuclear magnetic resonance spectra of tumors in anesthetized animals were obtained at 2.35 Tesla using surface coil localization. Areas under resonance peaks were normalized to the area under the peak of a phosphorus standard positioned at a fixed distance on the opposite side of the surface coil. Tumor concentrations of phosphomonoesters and phosphodiesters showed no change after tumor light doses up to 3000 J. Phosphocreatine, alpha-adenosine triphosphate (ATP), beta-ATP, and gamma-ATP signals decreased and inorganic phosphate signals increased with increasing light doses. The intratumor pH did not change significantly at these short times after PDT. In other R3327-AT and R3327-H tumor-bearing animals, [3H]misonidazole was administered 30 minutes prior to PDT treatments of both tumors. Twenty-four hours later, the tumors were resected in toto, and levels of retained [3H]misonidazole were determined in lased tumor specimens by liquid scintillation procedures. The amount of [3H]misonidazole activity in tumor tissue (covalently bound after hypoxic reduction) increased with light doses up to 3000 J. Sensitizer-adduct formation was found to correlate with the ratio of the concentration of inorganic phosphate to that of beta-ATP, both of which are presumed measures of tumor oxygenation status. These measurements have high-lighted the heterogenous nature of the oxygenation status of these experimental tumors. The precision of each assay for estimating tumor oxygenation is discussed.

Metabolism-induced binding of misonidazole was shown previously to be enhanced in hypoxic regions of V79 spheroids. Data are presented to show that the binding of misonidazole to EMT6 spheroids is similar to V79 in some respects and different in others. The binding rate in nitrogen was constant in all cells in EMT6 spheroids, whereas a 5-fold rise was found in binding rate across the outer 100 micrometers of V79 spheroids. A small enhancement of binding to the cells near the necrotic center in EMT6 spheroids in air was found, which was similar to the binding to chronically hypoxic cells in aerobic V79 spheroids. The binding rate in 1--5% oxygen was similar in EMT6 and V79 spheroids. Both showed a large rise in binding rate across the outer 120 micrometers and a constant binding rate to healthy cells interior to this. Since 1.5 oxygen should supply only the outer 40 micrometers of a typical spheroid, something beyond a simple oxygen diffusion limitation is required to explain the data. One possible explanation is the diffusion of some of the reactive product away from the hypoxic cells which produce it. This was tested by incubating a single cell suspension in misonidazole at 0.5% oxygen in the presence of absence of V79 spheroids. No evidence was found for an appreciable loss of reactive product from the spheroids.

Initial energy utilization in the twitch is visualized as the result of the activity of two distinct processes. The first is the calcium-pumping activity of the sarcoplasmic reticulum, which has a constant energy requirement under normal conditions. The second is the chemomechanical transduction process consisting of a variable number of quantal contractile events, each with a fixed enthalpy equal to the molecular enthalpy of adenosine triphosphate (ATP) hydrolysis in vivo. This enthalpy appears either as heat or as contractile element work. Total enthalpy varies according to the number of quantal contractile events that occur in the twitch cycle. The basis of the variation is suggested to be velocity-dependent activity of the actomyosin ATPase, allowing more quantal events to occur in a contraction cycle when shortening occurs. The classical designation "activation heat" is held to be appropriate for the first process. The partition of the enthalpy of the second process that is currently in vogue is held to be misleading and a new formulation is suggested in which the properties of the quantal contractile event are reflected in general terms. The formulation of the proposed transduction model represents a conceptual return to the viscoelastic theory, but at a quantal level. The model can explain the results of the preceding paper and is adaptable to different muscles without having to postulate fundamental differences in energy utilization.

The effects of altering the concentrations of sodium (Na + ) and calcium (Ca 2+ ) ions on the isometric energy output of rabbit papillary muscle at room temperature were examined by a myothermic technique. The well-known inotropic changes resulting from these alterations were not correlated with parallel changes in the magnitude of the tension-independent heat. Instead, the magnitude of the tension-independent heat was directly correlated with both increasing (Na + ) concentration and with increasing (Ca 2+ ) concentration, and these effects were additive. The energetics of ions pumping in cardiac muscle are discussed quantitatively and it is suggested that the combined enthalpy consumption of both the (Na + ) and the (Ca 2+ ) pumps constitutes the major determinant of cardiac tension-independent heat production.

In recent years, immersive virtual reality has been used in disciplines such as engineering, sciences, and education. Point-cloud technologies such as laser scanning and unmanned aerial systems have become important for creating virtual environments. This paper discusses creating virtual environments from 3D point-cloud data suitable for immersive and interactive virtual reality. Both laser scanning and sUAS point-clouds are utilised. These point-clouds are merged using a custom-made algorithm that identifies data gaps in the master dataset (laser scanner) and fills them with data from a slave dataset (sUAS) resulting in a more complete dataset that is used for terrain modelling and 3D modelling of objects. The terrain and 3D objects are then textured with custom-made and free textures to provide a sense of realism in the objects. The created virtual environment is a digital copy of a part of the Penn State Wilkes-Barre campus. This virtual environment will be used in immersive and interactive surveying laboratories to assess the role of immersive virtual reality in surveying engineering education.

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.

Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. Cell viability was measured microscopically with trypan blue staining. Cell survival after various doses was computer-fitted to either a simple linear or a linear-quadratic equation. Twenty-four hours after a 10-Gy dose of 137Cs gamma rays, DNA fragmentation to nucleosome multimers was strongly expressed in only DU-145 cells. In this cell line, when centrifugation at 12,000g for 10 min was used to separate fragmented from large molecular weight DNA, the proportion of DNA in the supernatant increased to a maximum of approximately 17% of the total by 10-12 h after radiation treatment. Cell death 24 h after irradiation measured by trypan blue exclusion assays followed single-hit kinetics up to 80 Gy. The proportion of cells which were labeled with Apoptag displayed single-hit kinetics and yielded the same inactivation coefficient as measured by trypan blue. Together, these data indicate that the rapid (24 h) inactivation of irradiated DU-145 cells results from apoptosis and accounts for about 5% of the single-hit killing measured by clonogenic assay. Temporal studies of radiation-induced killing of DU-145 cells distinguished this rapid mechanism of cell death from the major mechanism (72-144 h). These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.

Seven second-generation hypoxic markers of the iodinated azomycin nucleoside class have been synthesized and tested for hypoxia marking activity with tumor cells in vitro and in vivo. β-D-lodoazomycin galactoside (IAZG) and β-D-iodoazomycin xylopyranoside (IAZXP) demonstrated superior hypoxia marking properties relative to IAZA because of their higher water solubilities, rapid plasma clearance rates from tumor-bearing mice and maximum tumor/blood (T/B) and tumor/muscle (T/M) ratios. Our studies with animal tumor models show that T/B or T/M ratios of these markers determined by scintigraphy or planar imaging can predict for the relative degree of tumor hypoxia and for tumor radioresistance. © 1997 John Wiley & Sons, Ltd.

When active transport is electrogenic in a tissue that is continuously active, such as cardiac muscle, the active transport current is as important in the generation of the action potential as are the passive currents. A thermodynamically constrained kinetic model of electrogenic active transport of sodium and potassium ions has been developed in which the influences of voltage and chemical composition are explicitly defined. This model is coupled to a system of passive permeabilities, of the minimum degree of complexity, to simulate the integrated activity of active and passive ion transport in the generation of the cardiac action potential. Results of preliminary simulations indicate that electrogenic active transport provides a mechanism for slowly changing currents both within the time scale of an action potential as well as of many action potentials. The presence of active transport also complicates the interpretation of isotopic flux measurements and the separation of currents.

Animation of human movement can be based either on analog inputs derived directly from actual movements or on symbolic inputs chosen to produce the desired movement. The former type of input can be quite accurate and objective but is a description of the required movement whereas the latter is often quite imprecise and subjective but provides an analysis of the required movements. Two existing systems for a computer based animation are being used to explore the problems involved in integrating such inputs. Specifically, animation driven by analog signals from electro-goniometers is integrated with animation derived from Labanotation commands; the results are illustrated with a short movie.

Specific monovalent cations control the modification of reovirus infectivity by chymotrypsin. Digestion in K(+), Rb(+), or Cs(+) reduces infectivity several logs, whereas in Na(+) or Li(+) digestion markedly enhances infectivity.

A technique using collagenase has been devised to release and separate, with reproducibility, hematopoietic cells (HC) from various microenvironments of mouse femurs. HC were assayed by an in vitro gel culture technique used traditionally to score granulocyte-macrophage precursor cells (CFU-C). CFU-C which resided in the medullary cavity and endosteal regions were sensitive to ionizing radiation and resistant to misonidazole (MISO) cytotoxicity. CFU-C which resided within the compact bone were resistant to ionizing radiation and sensitive to the cytotoxic action of MISO. These results suggest that HC which reside in the bone are hypoxic and retain clonogenic potential. When animals were exposed to various treatments with MISO followed by myelotoxic doses of cyclophosphamide (CTX) or total body irradiation (TBI), the LD50 of both agents was significantly reduced. This result suggests that a hypoxic component of HC could be important in the regenerative process within the marrow after such myelotoxic trauma.

Tolyporphin (TP), a porphyrin extracted from cyanobacteria, was found to be a very potent photosensitizer of EMT-6 tumor cells grown both in vitro as suspensions or monolayers and in vivo in tumors implanted on the backs of C.B17/Icr severe combined immunodeficient mice. Thus, during photodynamic treatment (PDT) of EMT-6 tumor cells in vitro, the photokilling effectiveness of TP measured as the product of the reciprocal of D50 (the light dose necessary to kill 50% of cells) and the concentration of TP is approximately 5000 times higher than that of Photofrin II (PII), the only PDT photosensitizer thus far approved for clinical trials. TP almost exclusively localizes in the perinuclear region and specifically in the endoplasmic reticulum (ER), as shown by microspectrofluorometry on single living EMT-6 cells costained with the ER and/or Golgi fluorescent vital probes, 3,3'-dihexyloxacarbocyanine iodide and N-[4,4-difluoro-(5,7-dimethyl-BODIPY)-1-pentanoyl]-D-erythro-sphin gosine (Molecular Probes, Eugene, OR). As a result, the singlet oxygen-mediated photodynamic activity of TP induces an effective inactivation of the acyl CoA:cholesterol-O-acyltransferase, a sensitive marker of ER membrane integrity and alterations of the nuclear membrane. In vivo, with the EMT-6 mouse tumor model, an exceptional effectiveness is also observed as compared to that of PII and other second generation photosensitizers of the pheophorbide class, which are themselves much more potent than PII. The outstanding PDT activity of TP observed in vivo may be due to its unique biodistribution properties, in particular much less extraction by the liver, resulting in a higher delivery to other tissues, including tumor.

Although a number of movement notation systems have been invented, only a few have been developed and are in substantial use. Movement notation systems are inherently complex and therefore are difficult to master. The system described here is designed to accept Labanotation commands as input when they are entered with a light pen at a computer graphics terminal, to interpret the commands and to display the output as a computer driven movie of dancing figures. To enhance the capabilities of the computer assisted notator a macro language is being developed to make more powerful commands available. The characteristics of the macro language and of a possible higher level language are discussed.

Animation of human movement can be based either on analog inputs derived directly from actual movements or on symbolic inputs chosen to produce the desired movement. The former type of input can be quite accurate and objective but is a description of the required movement whereas the latter is often quite imprecise and subjective but provides an analysis of the required movements. Two existing systems for a computer based animation are being used to explore the problems involved in integrating such inputs. Specifically, animation driven by analog signals from electro-goniometers is integrated with animation derived from Labanotation commands; the results are illustrated with a short movie.

Monte-Carlo computer simulation of random strand breakage has been applied to analysis of alkaline sucrose gradient sedimentation profiles from irradiated DNA. The method is simple and widely applicable, is valid at all radiation doses and for all size distributions of the unirradiated DNA. The data is efficiently utilized, resulting in improved accuracy in determining the efficiency of strand breakage. The analysis provides two tests of conformity of experimental data to the expected breakage and sedimentation behavior.

A combined myothermic and fluorescent investigation into the effects of substrate and calcium ions on the contractility of paired rabbit papillary muscles has shown that, (i) as shown in earlier studies pyruvate, when substituted for glucose, increases contractility, increases the levels of fluorescence and resting heat production and speeds the kinetics of fluorescent transients and the time cours of heat production, (ii) reducing the extracellular calcium level from 2.5mm to 1.25mm, decreases contractility without altering the resting fluorescence level and without changing the kinetics of fluorescent transients or the time course of active heat production. Neither treatment (substrate or calcium) altered the isometric heat coefficient. Evidence is presented showing that the fluorescence-time integral is linearly related to the energy expended in a cardiac contraction.

The sensitivity of intracerebellar nuclei neurones to pulse applications of l-aspartate. l-glutamate, N-methyl-d,l-aspartate and quisqualate was tested in rat cerebellar slices mainlained in vitro. The responses ol the nuclear neurones to the four agonists consisted of a transient and dose-dependent increase in their firing of simple spikes. When suprathreshold currents were used, quisqualate induced the highest increase in the spike discharge frequency of the cells. Quisqualate mediated responses were unaffected by steady applications of 2-amino-5-phosphonovalerate. whereas the sensitivity of the responses induced by the three other agonists was in the order N-methyl-d,l-aspartate. l-aspartate. l-glutamate. When the supervising solution was devoid of Mg2+ ions. N-methyl-d,l-maspartate and l-aspartate mediated responses were much potentiated, while quisqualate induced responses were not enhanced. In such a medium, l-glutamate elicited responses were more or less potentiated depending on cells.These results suggest that rat intraeerebellar nuclei neurones hear both N-methyl-d-aspartate and non-N-methyl-d-maspartate. probably quisqualate. receptors, and that laspartate and l-glutamate have a mixed action upon both types. l-Aspartate preferentially activates N-methyl-d-aspartate receptors, whereas l-glutamate predominantly acts via non-N-methyl-d-aspartate receptors, Furihermore, the potency of l-glutamate in activating N-methyl-d-aspartate receptors appears to vary as a function of the cells.

The effect of photodynamic therapy (PDT) on tumour perfusion in both anaplastic (R3327-AT) and well differentiated (R3327-H) Dunning prostatic tumours was studied using the radiopharmaceutical 99Technetium hexamethylpropyleneamine oxime (99mTc-HMPAO). Tumours in the left flanks of rats (Copenhage x Fischer, F1 hybrids) were treated with interstitial PDT when their volumes reached 2-3 cm3. Qualitative and quantitative data from pre- and post-PDT scintigraphy revealed a light-dose-dependent shut-down of tumour perfusion which was also time-dependent. Maximal shut-down, following a 1,600 J light-dose, occurred about 8 h post-PDT. Light exposure 2 h after the intravenous administration of the photosensitiser (Photofrin II) produced a greater vascular shut-down than did light exposure 24 h after the administration of the drug. Regional differences in perfusion within treated and non-treated tumours were measured by tomographic procedures. Light-dose-dependent volumes of perfusion shut-down were demonstrated in addition to the naturally occurring regional differences in tumour perfusion. This radiopharmaceutical may have future utility for monitoring the clinical treatment of solid tumours with PDT.

Summary The electrical and mechanical responses of the guinea‐pig ureter to raised extracellular potassium ion concentrations hove been studied. The response consisted of an initial series of rapid contractions associated with action potentials followed by the development of maintained tension associated with maintained depolarization. Relaxation and repolarization occurred when normal potassium ion concentration was restored. The threshold potassium ion concentration for the development of maintained tension varied between 25 and 50 mM. The amplitude of the maintained tension could be immediately and reversibly changed by varying the external calcium ion concentration, though the calcium ion concentration at which these changes appeared varied considerably between preparations. Absence or reduction of the initial contractions in low calcium ion solutions was always accompanied by absence or abnormality of the action potential. It is suggested that the maintained tension depends on an increased influx of extracellular calcium ions during the potassium ion‐induced depolarization. Although this mechanism is not excluded as a possible basis for contraction initiated by the depolarization of the action potential, it is likely that a calcium ions “store” of unknown nature is involved in the normal excitation‐contraction coupling process.

Both anaplastic and well-differentiated Dunning prostate adenocarcinomas were illuminated in anesthetized Fischer X Copenhagen rats by single-fiber and multiple-fiber illuminators. Each illuminator consisted of a 2-cm laterally diffusing optical fiber placed within a plastic brachytherapy needle which was implanted into a tumor. Light attenuation coefficients for various wavelengths were obtained from measures of the radial falloff of intensity with distance from single fibers. These coefficients served as input to a 2-dimensional (2-D) photodosimetry computer code which calculated relative light intensities in planes perpendicular to single-fiber and various multiple-fiber configurations. These calculations assumed uniform optical property of tissue throughout each tumor, uniform and equal illuminance from diffusing fibers, and precise needle implantation. Relative light intensities along specific tumor tracks were measured and compared with those predicted by the 2-D photodosimetry code. Agreement within +/- 14% was observed for all configurations studied. Variations in relative intensity in tumor planes perpendicular to a standard seven-fiber illuminator were determined as a function of the distance between the implanted needles. Light wavelengths of 700 nm and greater produced relatively uniform light fields (approximately +/- 20%) in the R3327-AT tumor with needle spacings of at least 1 cm. The addition of two fibers at the periphery of this illuminator (a nine-fiber illuminator) improved the uniformity of light delivery to the encompassed tumor volumes. The importance of precision photodosimetry for interstitial applications of photodynamic therapy is discussed.

Pheophorbide a prepared from the algae Spirulina was derivatized at the C(7)-carboxylic group by linking amino alkyls of various lengths and terminal functional groups. The compounds were purified by thin-layer chromatography (TLC) and by high-pressure liquid chromatography (HPLC). Solubilization of compounds by serum lipoproteins, the kinetics of compound uptake into mammalian cells, and photosensitizing effectiveness when activated by 673 nm laser light have been studied. Optimal photosensitizer uptake into cells and the greatest photosensitizing activity were observed with compounds having side-chain lengths of 4-6 carbon atoms which terminated in -OH and -CH3 groups. The most effective compounds were 3 orders of magnitude more potent than Photofrin in the degree of photoinactivation of cultured EMT-6 tumor cells. HDL and LDL significantly promoted the efflux of these photosensitizing drugs from cells, suggesting that their long-term retention in normal tissues in vivo would be minimal and produce little phototoxicity.

Purpose: Mammalian cells at mitosis contain chromatin in compacted form and are hypersensitive to ionizing radiation. Previous research had shown some chemicals that induce chromatin compaction within interphase cells act as radiosensitizers. Of these agents, cantharidin (LS‐1), which is an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), showed good radiosensitizing activity at non‐toxic doses. Cantharidin and 13 additional structural analogues (LS‐2–14) were tested for their radiosensitizing activity on tumour cells in vitro.Materials and methods: Twelve of the 14 cantharidin analogues were synthesized in the authors' laboratory. Various concentrations of the drugs were screened for toxicity and radiosensitizing effectiveness with asynchronous DU‐145 (human prostate carcinoma) cells. More detailed radiobiological studies of the more potent agents were performed with HT‐29 (human colon carcinoma) cells since they could be readily synchronized. The radiosensitization of G1 phase HT‐29 cells was measured after a 2‐h exposure to the more potent drugs and reductions of the surviving fraction after an acute dose of 2 Gy (SF2Gy) served to estimate their relative effectiveness. The increase in phosphorylation of histone 1 (H1) and histone 3 (H3) induced by these drug exposures was measured by Western blotting of protein extracts. Drug‐induced change in chromatin morphology was visualized by electron microscopy, and the alkaline comet assay (which measures DNA single‐strand breaks) was employed to measure the radiation sensitivity of cellular chromatin in the drug‐treated cells.Results: Of the 14 cantharidin analogues tested, LS‐1, LS‐2 and LS‐5 at concentrations of 3–20 µM showed little or no toxicity, produced elevated levels of H1 and H3 phosphorylation, and effected significant radiosensitization at low radiation dose. The chromatin in tumour cells treated with LS‐5 became visibly compacted and its DNA was about 1.6 times more sensitive to radiation‐induced strand breakage relative to that of control cells.Conclusions: The results confirm the authors' earlier studies that showed an increase in tumour cell intrinsic radiosensitivity by exposure to agents that promote chromatin compaction. LS‐5 was identified as the optimal radiosensitizing agent of this class of compounds. Radiosensitization was correlated with chromatin compaction and elevated phosphorylation of H1 and H3. The DNA in drug‐treated cells exhibited an enhanced sensitivity to radiation‐induced single‐strand breakage.

At one hour after injection of 100,μc of C14-formate into mice with advanced Ak-4 leukemia, it has been shown by means of blood smear autoradiograms that about 90% of the leukemic cells are highly active as compared to about 5% of the more mature lymphoid elements. No active polymorphonuclears, erythrocytes, or thrombocytes were observed within one hour.

Purpose : To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day −1 fractions and to low dose-rate brachytherapy. Materials and methods : Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the α- and β-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of β-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. Results : The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the β-mechanism. α-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high α-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Conclusions : Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (α) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of β-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

SummaryThe temperature dependence of DNA degradation has been characterized in E. coli Bs−1 and an activation energy of 17·2 kcal/mole determined for the enzyme function between 20°c and 40°c. The maximum rate of the breakdown function was measured to be near 42°c. At temperatures greater than 45°c heat inactivation of the enzyme system in vivo is evidenced.The dependence of DNA degradation at 37°c on radiation dose has been measured. The radiation appears to produce substrate for the degradative enzyme system, which is present in the cells as normal constituents of the cells. The Michaelis rate constant for such an enzyme reaction would be equivalent to a substrate concentration produced by 8·5 krads irradiation.The buoyant density of DNA throughout the degradative process was measured and found to be the same as double-stranded DNA from unirradiated cells. Models involving the in vitro mechanisms of actions of exonucleases I, II and III predict that a single-stranded component of the DNA should exist. This single-stranded component has not been observed.

ARTICLESPotentiating effect of potassium on skeletal muscle twitchJB ChapmanJB ChapmanPublished Online:01 Sep 1969https://doi.org/10.1152/ajplegacy.1969.217.3.898MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByK+-induced twitch potentiation is not due to longer action potentialCraig Yensen, Wadih Matar, and Jean-Marc Renaud1 July 2002 | American Journal of Physiology-Cell Physiology, Vol. 283, No. 1The Role of the Sarcolemma Action Potential in FatigueThe effect of K+ on the recovery of the twitch and tetanic force following fatigue in the sartorius muscle of the frog, Rana pipiensJournal of Muscle Research and Cell Motility, Vol. 15, No. 4Some antagonists of dantrolene sodium on the isolated diaphragm muscle of the rat12 April 2011 | Journal of Pharmacy and Pharmacology, Vol. 29, No. 1A COMPARISON OF THE EFFECTS OF SODIUM THIOCYANATE AND DANTROLENE SODIUM ON A MAMMALIAN ISOLATED SKELETAL MUSCLE19 July 2012 | British Journal of Pharmacology, Vol. 59, No. 1Work-induced potassium changes in skeletal muscle and effluent venous blood assessed by liquid ion-exchanger microelectrodesPflügers Archiv European Journal of Physiology, Vol. 362, No. 1Effect ofP CO2, bicarbonate and lactate on the isometric contractions of isolated soleus muscle of the ratPfl�gers Archiv European Journal of Physiology, Vol. 320, No. 2 More from this issue > Volume 217Issue 3September 1969Pages 898-902 Copyright & PermissionsCopyright © 1969 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1969.217.3.898PubMed5807718History Published online 1 September 1969 Published in print 1 September 1969 Metrics

Recently, the National Institutes of Health have identified functional imaging as an important focus for research. In addition, the number of markers available for measuring tumor genotype and phenotype is rapidly increasing, so that the biologic basis of several imaging procedures can be

Mammalian cells at mitosis, differentiated lymphocytes, and some radiation-hypersensitive mutants in interphase contain all or a measurable portion of their chromatin in condensed/compacted form and are hypersensitive to ionizing radiation by the mechanism described by single-hit inactivation kinetics (alpha). These observations led to the investigation as to whether compacted chromatin in interphase is the target that determines the widely variable alpha-parameters and surviving fractions of 2 Gy (SF2Gy) measured for human tumor cell lines. Six cell lines whose SF2Gy ranged from 0.29 to 0.73 were used for this study. Their different radiosensitivities were associated mainly with differences in their single-hit inactivation parameters (alpha). Electron microscope images of interphase nuclei were optically scanned, and the pixel densities were digitized for quantitative analyses. A significant correlation between the percentage of nuclear pixels with densities similar to those found in mitotic chromosomes (percent compacted chromatin) and the alpha-inactivation parameters was observed. Digital analyses of electron and/or confocal microscope images of chromatin in interphase tumor cells in biopsy specimens could become a rapid assay for predicting the intrinsic radiosensitivity of tumor clonogens. This research has also identified some inhibitors of protein (histone) phosphatases that promote chromatin compaction and radiosensitize cells to 2-Gy dose fractions.

A previous paper described a kinetic model for electrogenic sodium-potassium transport in cardiac muscle, combining a thermodynamically-constrained transport model with simple passive permeabilities for sodium and potassium to generate a cardiac action potential (Chapman, Kootsey & Johnson, 1979). The present paper explores the extent to which this simplest of active-passive transport models can account (without further modification) for the electrophysiological behavior of cardiac muscle. The long term (several minutes) changes in the duration of the action potential observed following a change in stimulation rate are predicted by the model through a shift in the steady-state current-voltage relationship caused by small changes in inside ion concentrations. The diastolic hyperpolarization observed following an increase in rate is also predicted, including the linear relationship between the maximum diastolic depolarization and the rate of stimulation. Varying the outside potassium concentration in the model produces changes in the rest potential and current-voltage relationship similar to published data. Deviations from ideal potassium electrode behavior occur at both high and low concentrations because of effects on the pump. The model not only predicts the observed shift of the current-voltage curve in the depolarizing direction with increasing [K+]0, but also the crossing of the curve in normal [K +]0 without having to assume a variation in gK. Anoxia was introduced into the model by changing the concentrations of ATP and ADP, thereby enabling the model to account for the rapid diastolic depolarization observed in myocardial ischemia.

In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity.Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion.The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF.The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.

The chain of reactions, resulting in mutation and cell death, initiated when ionizing radiation interacts with mammalian cells is complex and traverses a time-scale from a fraction of a picosecond to a few hours. Recently, progress has been made in identifying some of the more important pathways and the potentially damaging free radical intermediates. Protection can, in principle, result from modification at several steps in these reaction chains, each with its distinctive time-scale. To date we have demonstrated that radioprotection can be effected in mammalian cells by the modification of processes at three quite different times. In this paper, the temporal sequence of radiation-initiated events are reviewed and the potential for or measurement of radioprotection of each is discussed. Such an understanding of cellular radioprotection has become possible by our improved understanding of cellular radiation mechanisms derived from recent studies of its chemistry [1–4].

Contractile energetics have been studied in twitches of toad sartorius muscle at 6-7 degrees C. Isometric and isotonic energy production has been measured and plotted against a wide range of developed tensions and tension-time integrals. These parameters were varied by altering the isotonic load or by changing the preset isometric length. The isometric tension-independent heat was 1.12 +/-0.18 (SD) mcal/g. The isometric heat coefficient Pl(0)/H was 12.0 +/-1.4 in muscles having twitch to tetanus ratios ranging from 0.4 to 0.6. Isometric enthalpy increased monotonically with tension or tension-time integral but the correlation between isometric heat and these parameters was poor. Isotonic enthalpy consumption was always higher than isometric enthalpy for any given tension or tension-time integral; however, isotonic heat production was consistently less than isometric heat production. The isotonic heat for the highest load (3 g) was not significantly different from the isometric tension-independent heat. Thus isotonic heat production first decreased and then increased with increasing tension or tension-time integral. In the discussion it is shown that the results conflict with all current interpretations of muscle energetics.

Experiments have been carried out which demonstrate that leukemic cells immobilized in a cellophane bag implanted in the abdominal cavity of a host mouse incorporate C14-formate much more rapidly than do normal lymphocytes.

Rodent tumour models have been the 'workhorse' for tumour oxygenation research and for investigating radiobiological hypoxic fraction. Because of the intertumour heterogeneity of blood flow and related parameters, most studies have pooled information derived from several different tumours to establish the statistical significance of specific measurements. But it is the oxygenation status of and its modulation in individual tumours that has important prognostic significance. In that regard, the bioreducible hypoxic marker technique was tested for its potential to quantify oxygenation changes within individual tumours. Beta-D-iodinated azomycin galactoside (IAZG) and beta-D-iodinated azomycin xylopyranoside (IAZXP) were each radiolabelled with Iodine-125 and iodine-131 for measurements of animal tumour oxygenation. The tumour-blood (T/B) ratio of marker radioactivity in mice after the renal excretion of unbound marker (at 3 h and longer times) had been shown to be proportional to radiobiological hypoxic fraction. When markers labelled with both radioisotopes were administered simultaneously to EMT-6 tumour-bearing scid mice, T/B ratios were found to vary by up to 300% between different tumours, with an average intratumour variation of only approximately 4%. When the markers were administered 2.5-3.0 h apart, changes in T/B ratios of 8-25% were observed in 10 out of 28 (36%) tumours. Changes to both higher and lower hypoxic fraction were observed, suggestive of acute or cycling hypoxia. When 0.8 mg g(-1) nicotinamide plus carbogen was administered to increase tumour oxygenation, reductions in T/B ratios (mean deltaT/B approximately 38%) were observed in all tumours. Similar results were obtained with Dunning rat prostate carcinomas growing in Fischer x Copenhagen rats whose T/B ratios of IAZG and radiobiological hypoxic fractions are significantly lower. These studies suggest that fluctuating hypoxia can account for at least 25% of the total hypoxic fraction in some tumours and that correlations between bioreducible marker avidity and related tumour properties will be optimal when the independent assays are performed over the same time period. This dual hypoxic marker technique should prove useful for investigating both spontaneous and induced oxygenation changes within individual rodent tumours.

The radioprotective action of DDC on normal hematopoietic tissue in mice was evaluated. An increase in CFU-S and CFU-GM in DDC treated unirradiated control mice was consistently observed. When post-irradiation survival of CFU-S and CFU-GM from DDC treated animals was normalized to account for this observed increase, protection factors ranging from 0.9 to 1.6 were observed. These protection factors are significantly lower than those reported by others. Maximum radioprotection was observed when irradiations were performed 15 to 30 min after DDC treatment; maximum increase in GFU-GM occurred within one to two hr following treatment. The increase in CFU-GM is analogous to that observed in animals treated with endotoxin, a non-thiol radioprotector. When C3H/HeJ mice, which are genetically incapable of responding to endotoxin, were challenged with DDC, an average CFU-GM increase of 1.7 times was observed, suggesting that the stimulatory effects of DDC were not due to endotoxin contamination. DDC was administered daily for three days before irradiation and little or no increase in CFU-GM and no radioprotection was observed, suggesting that the marrow can become refractory to DDC. When WR-2721 was tested in similar studies, a dose-modifying radioprotection was observed, with no significant non-specific stimulation of hematopoietic cells.

Summary Highly lipophilic and proteinbound derivatives of l-(2-phenoxyethyl)-2-nitroimidazole (PENI; RGW609), a known radiosensitizer, have been prepared by introducing one (IPENI) and two (DIPENI) iodine atoms into the phenyl ring via electrophilic substitution. The electron affinity of all 3 compounds, as determined by differential pulse polarography, was similar to that for MISO, a radiosensitizer which has undergone clinical trial, but P values were 2-3 orders of magnitude greater that for MISO and %PB values were as high as 30% in vitro compared to less than 1% for MISO. 131I-PENI was prepared by catalysed halogen exchange with Na131I in greater than 90% yield, and was found to be chemically and radiochemically stable in solution for at least 2 weeks. Whole-body studies in BDF/1 mice bearing EMT-6 tumors showed rapid hepatic extraction and biliary elimination with little or no accumulation in any other tissue including tumor and fat, indicating that P and %PB values had little impact on in vivo distribution and disposition. 131I-PENI was not measurably deiodinated in vivo. IPENI and DIPENI are radiosensitizers by inference only, that is, they have not been tested for radiosensitizer properties. However, PENI, the parent compound, has been shown to be active as a radiosensitizer, and the peak potentials (polarographic) for both IPENI and DIPENI fall near those of MISO, PENI and most other 2-nitroimidazole sensitizers. The low levels of concentration in target tissues achieved by 131I-PENI mitigate against its use as a diagnostic agent. Metabolic and pharmacokinetic studies, as well as an investigation of their radiosensitizing properties are required to determine whether IPENI or DIPENI have the potential to be used therapeutically.

The voltage-dependent rate constants of the Hodgkin-Huxley equations for the n, m, and h systems are tested for consistency with the Second Law of Thermodynamics as applied to the kinetic behaviour of independent particles moving in a closed thermodynamic system according to first-order rate laws. No consistency with Boltzmann distribution theory and classical thermodynamics is found for any of the n, m, or h rate constants without the additional proviso that the equivalent valencies of the n, m, and h particles are also voltage-dependent. A model is provided showing how non-linear membrane potential profiles could create an apparent voltage-dependent variation in the equivalent valency of particles experiencing only a fraction of the electric field. There is an unexplained discrepancy between the classical description of the m system and the more recent description of it in association with measurement of gating currents: the classical description requires voltage-dependent valency for thermodynamic consistency whereas the recent description is a thermodynamically formulated one that assumes constant valency independent of voltage for the independent m particles. The formulations presented become void if departures from first order independent behaviour are allowed.

Reovirus, an animal virus whosegenome consists ofdouble-stranded ribonucleic acid(RNA), hasbeenshowntocarryan RNA polymerase (transcriptase) inthecoreofthevirion (2,11,13). Thepolymerase activity canbedemonstrated in vitro after thevirions havebeentreated either to remove ortodamage theouterprotein coatand exposethecoretothesubstrates. During studies on theproperties ofthereovirus-core RNA polymerase invitro, thepresenceofa nucleoside triphosphate phosphohydrolase activity inthe purified viral coreswas detected. Thisactivity converts thenucleoside triphosphates (NTP)to nucleoside diphosphates (NDP)invitro. Inthis communication, some properties ofthis enzyme arepresented withevidence whichindicates that itisanintegral portion ofthevirion cores.

The recent advances in LiDAR (Light Detection and Ranging) have allowed for the remote sensing of important forest characteristics to be more reliable and commercially available. Studies have shown that this technology can accurately estimate forest characteristics including individual tree location, canopy height, and crown diameter. These values are used to estimate biophysical properties of forests such as basal area and stand volume. This study assesses the accuracy of LiDAR-derived estimates of forest characteristics including diameter and height against traditional timber cruising through field sampling. A commercially available program, TiFFS (Toolbox for LiDAR Data Filtering and Forest Studies), is used for comparison. Results are discussed with the focus on forestry operation.  MCFNS 2(2):145-152.

The purpose of this study was to determine, with a rodent tumor model, if microelectrode measurements of unmodulated tumor oxygenation predict for the avidity of hypoxic markers to tumor tissue.The rapidly growing, anaplastic variant of the Dunning rat prostate carcinoma cell line (R3327-AT) was implanted subcutaneously on the upper backs of Fischer X Copenhagen rats. Approximately 100 measurements of PO2 were obtained from tumors of 5-10 g in animals that were restrained and then subjected to different anesthetic procedures. Values of median PO2 (in mm Hg) and percentage of measurements <5 mm Hg obtained from individual tumors were used to define tumor oxygenation status. The radiodiagnostic hypoxic markers beta-D-iodinated azomycin galactopyranoside (IAZGP) and [99mTc]HL-91 were simultaneously administered to 26 animals whose tumor oxygen levels had been measured. Six hours after marker administration, the animals were killed; tumor, blood, and muscle tissues were sampled; and percentage injected dose per gram (%ID/g*), tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) parameters were determined. Parameters of marker avidity to individual tumors were linearly correlated with microelectrode measurements of tumor oxygenation to determine the significance of inverse associations.The median PO2 of 41 tumors varied from 2.0 to 20.9 mm Hg, with an average value of 7.5 +/- 1.4 mm Hg. Six tumors had unusually high values; that is, >10 mm Hg, and when these were excluded from the analysis, the average median PO2 of the remaining 35 was 4.3 +/- 0.7 mm Hg. When electrode measurements of tumor oxygenation were obtained under conditions of halothane anesthesia with the animals breathing O2, carbogen, or air, median PO2 values increased significantly (P = 0.001). When animals were deeply anesthetized by intraperitoneal injection of ketamine-xylazine, median PO2 values were not significantly different (P = 0.13) from those obtained while the animals were restrained and breathing air. There was no inverse correlation of significance between the electrode measurements of median PO2 and the avidity of beta-D-IAZGP nor [99mTc]HL-91 in this tumor model. The range of median PO2 values in these tumors was at least 3 mm Hg, and the range of hypoxic marker avidity was less than twofold.These data demonstrate that microelectrode measurements of rat tumor oxygenation did not correlate with the avidity of the two hypoxic markers, at least in this tumor model. The larger dynamic range of tumor oxygen measurements obtained with microelectrodes might be biased to low values by their necrotic fractions, the zones within solid tumors that contain dead cells and debris that will not be labeled by bioreducible hypoxic markers. Hypoxic marker avidity to individual tumors will have to be validated by other assays that can predict for their radiosensitivity.

The cyclam ligand (1,4,8,11-tetraazacyclotetradecane) was condensed with various azomycin-containing synthons to produce chemical compounds that could chelate radioactive metals. It was expected that these radiolabeled markers would become bound selectively to hypoxic cells on the bioreduction of their azomycin substituent.The markers were radiolabeled with (99m)Tc, (67)Cu, or (64)Cu. Their uptake and binding to tumor cells in vitro was characterized as a function of time and oxygen concentration. These data defined the hypoxia-specific factor, the ratio of the initial rate of marker binding to severely hypoxic relative to aerobic cells. In addition, the concentration of oxygen (in the equilibrium gas phase) that inhibited binding to 50% of the maximum rate was determined. The in vivo biodistribution and clearance kinetics of the favorable markers were investigated with severe combined immune deficiency mice bearing EMT-6 tumors whose radiobiologic hypoxic fraction (RHF) was approximately 40%. The specific activity (percentage injected dose per gram [%ID/g]) in normal and tumor tissue and the tumor-to-blood and tumor-to-muscle ratios of the optimal markers were also measured for Dunning prostate carcinomas of anaplastic (RHF = 15%-20%) and well-differentiated (RHF < 1%) histology growing in Fischer X Copenhagen rats. Planar images were acquired with some markers from these tumor-bearing rats.The tumor uptake of these cyclam-based markers is approximately 10 times higher when they are labeled with copper isotopes than when labeled with (99m)Tc. FC-327 and FC-334, di-azomycin-substituted cyclams, exhibited hypoxia-specific factors > or = 7.0. The oxygen concentration that inhibited their binding to 50% of the maximal rate was approximately 0.5% O(2), similar to that of the radiobiologic oxygen effect. The %ID/g of (64)Cu-FC-334 retained in EMT-6 tumors in mice and in the anaplastic and well-differentiated prostate tumors in rats 6 h after administration was approximately 6.5, 0.4, and 0.1, respectively. Marker activity in tumor was always less than that in liver and kidney. The tumor-to-blood and tumor-to-muscle ratios of (64)Cu-FC-327 and (64)Cu-FC-334 activity in R3327-AT tumor-bearing rats are higher than those observed for (64)Cu-di-acetyl-bis (N(4)-methylthiosemicarbazone) and approach those of beta-D-(125)I-iodinated azomycin galactopyranoside, the optimal hypoxia marker of the azomycin-nucleoside class.These data suggest that some azomycin-cyclams exhibit good hypoxia-marking potential to tumor cells in vitro and to animal tumors of known RHF. Both PET and SPECT could be used to image tumor hypoxia with markers labeled with (64)Cu and (67)Cu, respectively.