Variation in normal and tumor tissue sensitivity of mice to ionizing radiation-induced DNA strand breaks in vivo.

The efficiency of DNA strand break formation in normal and tumor tissues of mice was measured using the technique of alkaline elution coupled with a microfluorometric determination of DNA. This methodology allowed measurement of the DNA strand breaks produced in tissues irradiated in vivo with doses of radiation comparable to those used in radiotherapy (i.e., 1.0 gray) without the necessity for the cells to be dividing and incorporating radioactive precursors to label the DNA. The results showed that substantial differences existed among various tissues in terms of the amount of DNA strand break damage produced for a given dose of radiation. Of the normal tissues, the most breaks were produced in bone marrow and the least were produced in gut. Furthermore, strand break production was relatively inefficient in the tumor compared to the normal tissues. The efficiency of DNA strand break formation measured in the cells from the tissues irradiated in vitro was much more uniform and considerably greater than that measured in vivo, suggesting that the normal tissues in the animal may be radiobiologically hypoxic.

Kinetics of DNA cross-linking in normal and neoplastic mouse tissues following treatment with cis-diamminedichloroplatinum(II) in vivo.

The formation and repair of cis-diamminedichloroplatinum(II) (cis-DDP)-induced DNA cross-links in cells from a number of different mouse tissues, both normal and neoplastic, were compared in three different populations of animals, tumor-free mice and mice bearing a transplanted fibrosarcoma (either FSa or NFSa) in their thighs. Groups of mice were given i.v. injections of 4-12-mg/kg doses of cis-DDP, and the amount of cis-DDP-induced DNA cross-linking was determined at different times after injection using an adaptation of the alkaline elution technique. The degree of cross-linking in each tissue was linearly related to the dose of cis-DDP at either 6 or 24 h after injection and varied significantly among the different tissues, with FSa, NFSa, kidney, and liver showing the highest level of cross-linking of the tissues studied. The relative contributions of DNA-interstrand and DNA-protein cross-links to the elution profiles were estimated by proteinase K (PK) digestion. At either 6 or 24 h after injection with cis-DDP, the rate of elution of the DNA was substantially increased by PK, indicating a large contribution of DNA-protein cross-links. This effect was observed in all tissues studied, although the proportion of PK-resistant lesions appeared to vary from tissue to tissue, liver and spleen showing a significantly lower proportion of DNA-interstrand to total cross-links than either of the tumors. For liver, virtually no interstrand cross-links could be detected after PK treatment. The kinetics of the repair of cis-DDP-induced DNA cross-linking in these tissues were also compared. In cells from tumor-free animals, the amount of total (DNA-interstrand plus DNA-protein) cross-linking increased gradually, reaching a maximum after about 6 h; however, little evidence of repair of these lesions was observed in any of these normal tissues. In fact, the degree of cross-linking tended to increase somewhat between 6 and 24 h after injection. The kinetics of cross-linking in cells isolated from the FSa tumor were very different; while there was an initial increase in cross-linking up to 6 h, these lesions were subsequently repaired, although at a somewhat slower rate than has been reported for cultured mammalian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Nitroimidazole (EF5) binding predicts radiation resistance in individual 9L s.c. tumors.

The presence of hypoxic tumor cells is known to be an important cause of radiation treatment resistance in vivo. The ability to predict the presence and extent of hypoxic cells in individual tumors would allow the addition of specific "antihypoxia"-based treatment regimes. Hypoxia can be monitored by measuring the binding of 2-nitroimidazoles. We have tested the hypothesis that binding of EF5, a fluorinated derivative of the 2-nitroimidazole, Etanidazole, can predict radioresistance in individual tumors. Fischer rats bearing 9L s.c. tumors were given injections i.v. with EF5 3 h before irradiation and tumor harvest. Tumor cells were dissociated for flow cytometric analysis and plating efficiency studies. EF5 binding was detected via monoclonal antibodies conjugated to the orange emitting dye, Cy3. In air breathing rats, for a given radiation dose, a large amount of variation in plating efficiency was seen. However, there was minimal variability of the plating efficiency for tumors irradiated in euthanized animals (hypoxic tumors; correlation coefficient for the fitted curve = 0.93) and in cells dissociated from tumors and irradiated in suspension (correlation coefficient for the fitted curve = 0.99), suggesting that varying sensitivity to the cell disaggregation technique was not responsible. In contrast, a good correlation between the relative radiation resistance or hypoxic survival and EF5 binding of "moderately" hypoxic cells in air breathing rats was identified using these techniques. In these 9L s.c. tumors, intertumor variation in oxygenation accounted for most of the range in individual tumor radiation response, and this was found to be independent of tumor size. This study provides evidence for the application of EF5 binding with monoclonal antibody detection as an in vivo predictive assay of individual tumor hypoxia and resultant therapy resistance.

Tamoxifen induces hypoxia in MCF-7 xenografts.

Tamoxifen is widely used as an adjunct therapy for breast cancer. We hypothesized that hypoxia develops in tumors as a result of tamoxifen treatment because tamoxifen has been reported to be antiangiogenic and thrombogenic. MCF-7 breast tumors were grown under estrogenic stimulation in 4-6-week-old CD-1 nu/nu female mice. When the tumors were approximately 5 mm in diameter, 17beta-estradiol pellets were replaced with either placebo or tamoxifen-containing pellets. Two days later, tissue oxygenation was measured using immunohistochemical detection of binding of the 2-nitroimidazole EF5. Intravascular oxygen partial pressures were measured noninvasively by oxygen-dependent quenching of phosphorescence of an injected dye that is excited by light pulses. Tamoxifen treatment increased hypoxia in the tumors, as measured by EF5 binding (P = 0.01 by Mann-Whitney test). This observation was not dependent on the presence of tamoxifen-induced necrosis. Intravascular oxygen partial pressures were lower in tumors relative to surrounding normal tissue in tamoxifen-treated tumors as compared to placebo-treated tumors. In vitro, tamoxifen did not modify the oxygen-dependent metabolism of EF5, indicating that the increased EF5 binding in tamoxifen-treated tumors reflects a physiological decrease in tissue oxygenation. The clinical significance of these observations is discussed in the context of the sequencing of tamoxifen with other therapies, and in light of recent data suggesting that hypoxia may be associated with genetic changes resulting in a more aggressive tumor phenotype.

Doppler ultrasound imaging detects changes in tumor perfusion during antivascular therapy associated with vascular anatomic alterations.

Noninvasive monitoring of antiangiogenic therapy was performed by serial power Doppler ultrasound imaging of murine tumors treated with recombinant interleukin 12, the results of which were correlated with assessments of tumor vascularity by microscopy. Growth of established K1735 tumors, but not of IFN-gamma-unresponsive K1735.N23 variants, was suppressed by treatment. Serial Doppler imaging of K1735 tumor vascularity during treatment revealed a progressive change from a diffuse perfusion pattern to a more punctate distribution. Quantitative analysis of the images revealed that color-weighted fractional average, representing overall tumor perfusion, consistently decreased in these tumors, primarily because of a decrease in fractional tumor cross-sectional area carrying blood flow. In contrast, these parameters increased in nonresponsive tumors during treatment. Confocal microscopy of thick tumor sections revealed a reduction in the density and arborization of vessels labeled in vivo by fluorochrome-conjugated lectin with effective treatment. Immunohistological examination of thin tumor sections confirmed the preferential loss of small vessels with successful therapy. Similar changes in tumor vascular anatomy and perfusion were also observed during recombinant interleukin 12 treatment of two other responsive murine tumor types. These results indicate that power Doppler ultrasound is a sensitive, noninvasive method for reporting functional consequences of therapy-induced vascular anatomical changes that can be used to serially monitor tumor perfusion and efficacy of antivascular therapy in clinical trials.

Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.

Detection of hypoxia in human squamous cell carcinoma by EF5 binding.

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.

Thermodynamic bookkeeping when nucleotides bind. Applications of the theory of linked functions.

The thermodynamic theory of linked functions was used to determine the numbers of modifier ions involved when nucleotides dissociate. Nucleotide dissociation constants, obtained spectrophotometrically using Dowex-1 resin as a model system, were plotted on log/log paper with respect to the modifier concentrations. The slopes of the lines represent the net number of modifier molecules/ions involved in the dissociation. Varying numbers of nucleotides are bound to the resin because the resin capacity is determined by the total number of charges bound. The nucleotides bind to the resin at comparable diffusion-limited rates, irrespective of how tightly they bind. When ATP binds at pH 6.8, 4 chlorides, 4 formates, 2 succinates or 1.4 citrates are displaced, indicating that the fully charged (ATP4-) nucleotide binds. By comparing ATP, ADP and AMP it was possible to evaluate the contributions of the adenosine moiety and each phosphate to the binding. Between pH 2 and 3, where ATP has two negative charges, ATP binds largely as the trianion, displacing 2.7 chlorides and 0.7 protons. In the presence of 4 mM magnesium, 0.58 magnesiums facilitate the dissociation by chelating 58% of the liberated ATP. Calcium behaved similarly to magnesium but aluminum, at pH 6.8, promoted the binding of ATP as an (A1.ATP)3- complex with the concomitant liberation of three chloride ions. These experimental thermodynamic stoichiometries were found to be independent of the concentrations of the other modifiers present. Thermodynamic linkage stoichiometries can be evaluated from log K vs. log (modifier) plots when a direct determination of modifier binding is impossible.(ABSTRACT TRUNCATED AT 250 WORDS)

Three solutions of the protein solubility problem.

Three simple equations are presented, which describe the variation of protein solubility (S) with changes in salt concentration, in terms of either the salt molality (M), the salt activity (ax), or the water activity (aw). Each equation yields, essentially independent, estimates of the numbers of salt ions (delta vx) and water molecules (delta vw) involved in the dissolution of a mol of the protein. The equations can be used to elucidate the physical significance of the parameters in other empirical equations for protein solubility.

Subunit dissociations in natural and recombinant hemoglobins.

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

Thermodynamic bookkeeping: a reinvestigation of proton and dicarboxylic acid binding to aspartate aminotransferase.

A reinvestigation of the effects of pH and salt concentration on the proton and dicarboxylic acid dissociation constants of pig heart aspartate aminotransferase shows that both anions and cations were involved concomitantly, both as stoichiometric reactants and bioenergetically. Equations are presented which can be used experimentally, to determine the numbers of salt ions (their thermodynamic stoichiometries) involved in biochemical equilibria such as proton and ligand dissociations from macromolecules. These equations were used to reinvestigate the effects of salts on the chromophoric pKa of the enzyme prosthetic group, the interaction of the enzyme with dicarboxylic acids, and the overall equilibrium for the transamination half-reaction.

Hypoxia and necrosis in rat 9L glioma and Morris 7777 hepatoma tumors: comparative measurements using EF5 binding and the Eppendorf needle electrode.

PURPOSE: The purpose of this study was to assess the presence of tumor hypoxia using two independent techniques: binding of the 2-nitroimidazole EF5 and Eppendorf needle electrode measurements. The distribution of tumor hypoxia was assessed with respect to tumor necrosis in corresponding histological studies. METHODS AND MATERIALS: Each of several rats bearing a subcutaneous 9L glioma or Morris 7777 hepatoma tumor was given EF5 i.v. to a final, whole-body concentration of 100 microM. About 2.5 h later, each rat was anesthetized, and needle electrode measurements were made in the tumor along 1-5 tracks (30-200 individual measurements). At 3 h post-EF5 injection, the tumor was excised and frozen. Frozen sections were analyzed for the presence and distribution of binding of EF5 and necrosis using immunohistochemical techniques followed by staining with hematoxylin and eosin (H&E). The histochemical analysis and electrode readings in similar regions of the tumor were compared. RESULTS: Electrode measurements were taken at 0.4-mm intervals along one-dimensional tracks, whereas EF5 binding measurements from tissue sections contained two-dimensional information at high spatial resolution ( approximately 2.5 micro). The EF5 measurements showed greater spatial heterogeneity than did the electrode measurements. In tumor regions with minimal necrosis, needle tracks with relatively high pO(2) readings were usually found to contain relatively low EF5 binding, and vice versa. Because EF5 binding is inversely related to tissue pO(2), this result was expected. The expected inverse correlation of the two techniques was most disparate in necrotic tumor regions (confirmed by H&E staining), where needle electrode measurements showed low to zero pO(2) values, but little or no EF5 binding was found. CONCLUSION: The two methods compared in this study operate in fundamentally different ways and provide substantially different information. EF5 binding provided detailed spatial information on the distribution of hypoxia in viable tumor tissue. There was no EF5 binding in necrotic tumor tissue because cells in such tissue were unable to metabolize the drug. In contrast, output from the needle electrode method appeared to represent a "track-average" tissue pO(2) and did not distinguish between extreme hypoxia and either macroscopic or microscopic necrosis. At the present time, the importance of tumor necrosis in determining treatment response is unknown. However, our data suggest that the Eppendorf needle electrode technique will overestimate the presence of hypoxia. Both techniques are potentially limited by sampling errors in tumors with heterogeneous distributions of hypoxia.

Hypoxia in human intraperitoneal and extremity sarcomas.

PURPOSE: The presence of hypoxia, measured by needle electrodes, has been shown to be associated with poor patient outcome in several human tumor types, including soft tissue sarcomas. The present report emphasizes the evaluation of hypoxia in soft tissue sarcomas based upon the binding of the 2-nitroimidazole drug EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide). EF5 has previously been shown to be predictive of radiation response in animal tumors and in in vitro studies. We have also previously reported studies of EF5 binding in human squamous cell tumors. Using fluorescent immunohistochemical techniques, we provide data on the presence and distribution of EF5 binding, as a surrogate for hypoxia, in human spindle cell tumors. METHODS AND MATERIALS: Patients with spindle cell tumors who were scheduled for tumor surgery were asked to participate in the Phase I trial of EF5. Approximately 48 h preoperatively, EF5 was administered i.v. at doses between 9 and 21 mg/kg. Binding in frozen sections of biopsied tissues was determined using monoclonal antibodies labeled with the green-excited, orange-emitting fluorescent dye, Cy3. Calibration studies were performed in vitro by incubating fresh tumor tissue cubes obtained from each patient with EF3 (an analog of EF5) under hypoxic conditions ("reference binding"). The goal of these calibration studies was to quantify the maximal binding levels possible in individual patient's tissues. The relationship between binding (in situ based on EF5 binding) and reference binding (in vitro based on EF3 binding) was determined. RESULTS: Eight patients were studied; 3 of these patients had gastrointestinal stromal tumors (GIST). The incubation of tumor tissue cubes in EF3 under hypoxic conditions demonstrated that all tumors bound drug to a similar extent. Reference binding showed a 3.2-fold variation in median fluorescence (113-356) on an absolute fluorescence scale, calibrated by a Cy3 dye standard. In situ binding in the brightest tumor section varied by a factor of 25.4 between the lowest and highest binding tumor (7.5-190.2). Heterogeneity of highest binding was greater between tumors than within individual tumors. A correspondence between EF5 binding and Eppendorf needle electrode studies was seen in the 5 patients with non-GISTs. CONCLUSION: Inter- and intratumoral heterogeneity of EF5 binding in spindle cell tumors has been documented. Patterns of binding consistent with diffusion limited hypoxia are present in human spindle cell neoplasms.

MIBG inhibits respiration: potential for radio- and hyperthermic sensitization.

INTRODUCTION: Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro. The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo. Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo. MATERIALS AND METHODS: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo. 31P-NMR was used to determine alterations in tumor cell pH in vivo. RESULTS: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro. The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro. Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. CONCLUSIONS: We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

Noninvasive detection of tumor hypoxia using the 2-nitroimidazole [18F]EF1.

UNLABELLED: The noninvasive assessment of tumor hypoxia in vivo is under active investigation because hypoxia has been shown to be an important prognostic factor for therapy resistance. Various nuclear medicine imaging modalities are being used, including PET imaging of 18F-containing compounds. In this study, we report the development of 18F-labeled EF1 for noninvasive imaging of hypoxia. EF1 is a 3-monofluoro analog of the well-characterized hypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetami de, which has been used to detect hypoxia in tumor and nontumor systems using immunohistochemical methods. METHODS: We have studied 2 rat tumor types: the hypoxic Morris 7777 (Q7) hepatoma and the oxic 9LF glioma tumor, each grown in subcutaneous sites. PET studies were performed using a pharmacological dose of nonradioactive carrier in addition to [18F]EF1 to optimize and assess drug biodistribution. After PET imaging of the tumor-bearing rats, tissues were obtained for gamma-counting of the 18F in various tissues and immunohistochemical detection of intracellular drug adducts in tumors. In one pair of tumors, Eppendorf needle electrode studies were performed. RESULTS: [18F]EF1 was excreted dominantly through the urinary tract. The tumor-to-muscle (T/M) ratio of [18F]EF1 in the Q7 tumors was 2.7 and 2.4 based on PET studies and 2.1, 2.5, and 3.0 based on gamma-counting of the tissues (n = 3). In contrast, the T/M ratio of [18F]EF1 in the 9LF glioma tumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and 1.4 based on gamma-counting of the tissues (n = 3). Immunohistochemical analysis of drug adducts for the two tumor types agreed with the radioactivity analysis. In the Q7 tumor, substantial heterogeneous binding was observed throughout the tumor, whereas in the 9LF tumor minimal binding was found. CONCLUSION: [18F]EF1 is an excellent radiotracer for noninvasive imaging of tumor hypoxia.

Aryl hydrocarbon hydroxylase induction in mitogen-stimulated lymphocytes by benzanthracene or cigarette tars adsorbed to asbestos fibers.

Human mitogen-stimulated lymphocytes, cultured in the presence of amosite asbestos (AS), demonstrated a slight increase in aryl hydrocarbon hydroxylase (AHH) activity compared with non-induced (control) cultures (P = 0.005). A much greater increase in enzyme activity occurred following addition of the inducers benzanthracene (BA) or cigarette tars (CT) to cell cultures (P less than 0.001 in both instances). Significant enzyme induction also occurred when AS fibers were first preincubated with CT or BA, washed with acetone, then added to lymphocyte cultures (P less than 0.003 in all instances). This increase in AHH activity was not as great, however, as the induction observed when BA or CT was added to cell cultures. No further increase in enzyme activity was noted when AS and CT or AS and BA were simultaneously added to culture lymphocytes (P greater than 0.070 in all instances). The results demonstrate that polycyclic aromatic hydrocarbons (PAH), such as BA and other components of CT, are adsorbed and transported by amosite AS particles. These AS-PAH complexes are capable of inducing AHH in cultured human lymphocytes.

Patterns of benzo[alpha]pyrene metabolism in normal human pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (PAMs) obtained by bronchopulmonary lavage from 6 normal non-smoking volunteers were incubated with [3H]-benzo[alpha]pyrene to ascertain the normal metabolism and conjugation of polycyclic aromatic hydrocarbons. Through the use of a crude glucuronidase preparation, both glucuronic acid and sulfate conjugates were examined. Phenols and quinones were identified by high-pressure liquid chromatography as the principal free metabolites formed during 1 h incubation with benzanthracene induced PAMs. In addition, phenols and quinones were major substrates utilized by these cells for conjugation during the incubation period. The ranges of benzo[alpha]pyrene metabolites produced by PAMs from non-smokers were compiled and the variation in production as well as detoxification of proximate carcinogenic benzo[alpha]pyrene metabolites are presented.

Effects of hyperglycemia on oxygenation, radiosensitivity and bioenergetic status of subcutaneous RIF-1 tumors.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supra-physiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17+/-1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO2 and 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays.

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality.

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.