Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo.

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.

The issue of risk in complex adaptive systems: the case of low-dose radiation induced cancer.

Living systems exist in hierarchical levels of biological organization, ascending from the basic atomic-molecular level, to the cellular level, the tissue-organ level, and the whole organism. All levels and elements at each level communicate with each other though intricate intra- and intercellular signaling through many specified molecular interactions. These regulate homeostasis between the system levels and their individual elements. The probability of a defined effect at the basic atomic-molecular level per impact increment of a toxic agent, such as ionizing radiation, at that level appears constant at low doses, even if the probability constant may change as a consequence of a previous exposure. Thus, at a given state of the system, the incidence of effect at the atomic-molecular level increases linearly with the number of impact increments in terms of energy deposition events. Primary effects may amplify to damage and there are immediate attempts at repairing the damage from an effect. Amplification and propagation of damage at, and from, the basic to higher levels of biological organization meets resistance, the degree of which per impact increment is not constant. It changes with the number of impact increments. This resistance encompasses both physico-chemical and biochemical reactions. The corresponding biochemical reactions express the physiological system's capacity to respond to perturbations of homeostasis at and between the various levels. Types and degrees of these responses depend on the system and the degree of homeostatic perturbation. At relatively mild to moderate degrees of perturbation, protective responses appear with a delay of hours and may last for months, shield also against endogenous non-radiogenic damage, and in doing so may prevail over radiogenic damage. With increasing degrees of homeostatic perturbation, damage eventually overwhelms adaptive protection. Thus, systems do not respond in a linear function of impact increments at the lowest level of biological organization. For assessing the probability of radiation damage per absorbed dose, i.e., risk, in complex adaptive systems, both damaging and protecting responses need attention, and to exclude one for the other is scientifically unjustified and misleading.

Preloading with L-tyrosine increases the uptake of boronophenylalanine in mouse melanoma cells.

To improve the effectiveness of boron neutron capture therapy, the possibility of stimulating boron uptake was investigated in an experimental model. B16F1 mouse melanoma cells were exposed to boronophenylalanine (BPA). The intracellular boron concentration followed Michaelis-Menten kinetics in the early incubation phase. In the late phase, cellular boron concentration was linearly related to the BPA concentration in the culture medium. Incubation with L-tyrosine before exposure to BPA (preloading) increased the intracellular boron concentration by a factor of three. It is concluded that in B16F1 cells BPA is transported by L and presumably ASC (alanine, serine, and cysteine) transport systems, and that boron uptake can be effectively stimulated by L-tyrosine preloading.

Selective delivery of liposome-associated cis-dichlorodiammineplatinum(II) by heat and its influence on tumor drug uptake and growth.

In an attempt to optimize the chemotherapeutic treatment of mouse tumor Sarcoma 180, liposomes containing cis-dichlorodiammineplatinum(II) (PDD), having transition temperatures of few degrees higher than the rectal temperature of mice, were used in combination with local hyperthermia. The uptake of radioactive PDD by tumors heated for 1 hr at 42 degrees was almost four-fold greater when the drug was associated in liposomes than if administered as free drug. Uptake of liposome-administered radioactive platinum by liver was twice that obtained with free PDD, whereas its incorporation by the kidney was the same by either method of drug administration. The effect of various combinations of hyperthermia, drug-containing liposomes, and free PDD on tumor growth was also studied. Treatment with liposome-associated PDD plus local heating resulted in a dose-modifying factor of 7 when compared with free drug and no hyperthermia. The dose-modifying factor was 2.5 when PDD liposomes and heat were compared within free drug and heat. Thus, PDD could be specifically released from liposomes by heat and resulted in both a greater drug uptake and a delayed tumor growth following treatment. Potential normal tissue toxicity problems, however, still need to be resolved before clinical application of this combined modality will be possible.

Influence of histone acetylation on the modification of cytoplasmic and nuclear proteins by ADP-ribosylation in response to free radicals.

Inhibition of histone deacetylase by addition of 5 mM n-sodium butyrate to the growth medium increases the utilization of [32P]NAD+ and ADP-ribosylation (ADPR) of total cellular proteins of V79, HeLa, mouse B16, mouse Fib/T and human T1 kidney cells by a factor of 1.2-2.3. When the ADP-ribosylase is challenged by exposing cells to damage by .OH radicals (25 microM CuSO4 2.8 mM H2O2) ADPR increases by factors of 5.7-6.0 and 3.2-4.0 in normal and butyrated cells, respectively. Operation of the free radical generator is supported by the response to EDTA and radical scavengers. Densitometric analysis of autoradiographs from SDS-gels show that butyrate exposure increases basal ADPR-modification of histones from T1 cells by factors of 1.1-1.9. Addition of .OH radicals increases the ADPR modifications of histones 4.4-8.7-fold in normal cells and 3.2-6.7-fold in butyrate exposed cells. Butyrate exposure elevates base level ADPR-modification and reduces subsequent ADPR-modification initiated by DNA damage. The results are consistent with the view that ADPR-modification and histone acetylation have overlapping functions and probably induce similar structural changes in chromatin.

Evidence for beneficial low level radiation effects and radiation hormesis.

Low doses in the mGy range cause a dual effect on cellular DNA. One is a relatively low probability of DNA damage per energy deposition event and increases in proportion to the dose. At background exposures this damage to DNA is orders of magnitude lower than that from endogenous sources, such as reactive oxygen species. The other effect at comparable doses is adaptive protection against DNA damage from many, mainly endogenous, sources, depending on cell type, species and metabolism. Adaptive protection causes DNA damage prevention and repair and immune stimulation. It develops with a delay of hours, may last for days to months, decreases steadily at doses above about 100 mGy to 200 mGy and is not observed any more after acute exposures of more than about 500 mGy. Radiation-induced apoptosis and terminal cell differentiation also occur at higher doses and add to protection by reducing genomic instability and the number of mutated cells in tissues. At low doses reduction of damage from endogenous sources by adaptive protection maybe equal to or outweigh radiogenic damage induction. Thus, the linear-no-threshold (LNT) hypothesis for cancer risk is scientifically unfounded and appears to be invalid in favour of a threshold or hormesis. This is consistent with data both from animal studies and human epidemiological observations on low-dose induced cancer. The LNT hypothesis should be abandoned and be replaced by a hypothesis that is scientifically justified and causes less unreasonable fear and unnecessary expenditure.

Physics must join with biology in better assessing risk from low-dose irradiation.

This review summarises the complex response of mammalian cells and tissues to low doses of ionising radiation. This thesis encompasses induction of DNA damage, and adaptive protection against both renewed damage and against propagation of damage from the basic level of biological organisation to the clinical expression of detriment. The induction of DNA damage at low radiation doses apparently is proportional to absorbed dose at the physical/chemical level. However, any propagation of such damage to higher levels of biological organisation inherently follows a sigmoid function. Moreover, low-dose-induced inhibition of damage propagation is not linear, but instead follows a dose-effect function typical for adaptive protection, after an initial rapid rise it disappears at doses higher than approximately 0.1-0.2 Gy to cells. The particular biological response duality at low radiation doses precludes the validity of the linear-no-threshold hypothesis in the attempt to relate absorbed dose to cancer. In fact, theory and observation support not only a lower cancer incidence than expected from the linear-no-threshold hypothesis, but also a reduction of spontaneously occurring cancer, a hormetic response, in the healthy individual.

Persisting radiation effect on murine 7-day and 12-day CFU-S.

If radiation-induced reduction in the proliferative ability of bone marrow cells is due to a change in the age structure of stem cells, it might be measurable by an increase in the ratio of 7- to 12-day spleen colonies. This increase has not been detected at either three weeks or one year after 5 Gy gamma-irradiation. It was found, however, that diameters of colonies and weights of hemopoietic tissue per colony were reduced after both periods of recovery. Thus, slow colony growth may have masked a change in the stem cell age structure. It is concluded that injury persists in a proportion of stem cells that continues the production of progeny and may contribute to late effects. Persisting cellular injury, in modified form, may affect all mitotically active tissues.

Repopulation ability and proliferation stimulus in the hematopoietic system of mice following gamma-irradiation.

The repopulation ability of bone marrow was measured following whole body gamma irradiation of mice. Immediately after irradiation (100--500 rad) bone marrow suspensions were transfused into lethally irradiated syngeneic recipients. At various time intervals after transfusion (1 hour--20 days) DNA synthetic activity in femur and spleen was measured by incorporation of 125iodo-deoxyuridine (125IUdR). The cellularity of femoral bone marrow and the spleen weight was determined. A dose dependent retardation of repopulation was found to be correlated with an increasing duration of an elevated proliferation index (125IUdR-incorporation per unit bone marrow cells of per unit spleen weight, resp.). This suggests that the organism exerted a stimulus on the hematopoietic system to promote regeneration. This proliferation stimulus was kept at a high level unit a certain degree of hematopoietic function was restored either by regeneration of the bone marrow or, by compensating proliferation, in the spleen. The repopulation ability apparently was not only impared by the loss of stem cells but also by the induction of genetic damage in stem cells whose survival and partial functioning may have been enabled by the elevated proliferation pressure.

Proliferation kinetics of early hemopoietic precursor cells with self sustaining capacity in the mouse, studied with 125-I-labeled iodo-deoxyuridine.

With a new labeling technique in radiation chimeras, an attempt was made to determine the duration of the phases of the stem cell cycle including shortest and mean generation time and to estimate the number of hemopoietic stem cells per unit of bone marrow cellularity. The DNA of bone marrow cells in DNA synthesis was labeled with 5-125I-2'-deoxyuridine. The labeled cells were followed after being transfused into fatally irradiated mice. The stem cells were found to have a half-time of about 4.3 days in the donor mice. The average time in the population, i.e. the turnover time of the stem cells, was 6.2 days. The half-time did not change significantly even after transfusion of bone marrow into lethally irradiated recipient mice. Tritiated thymidine (3H-TdR) suicide technique revealed that bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients behaved differently--S-phase in cells seeding to femurs being shorter. The radiosensitivity of stem cells in S-phase had a D0 of 80 rad whereas stem cells distributed throughout the whole cell cycle had a D0 of 185 rad. The respective extrapolation numbers were 1.23 and 1.14. It is calculated that 2--7% of all nucleated bone marrow cells belong to self renewing stem cell populations. The method described provides a new approach for the study of hematological stem cells.

An assay for the measurement of residual damage of murine hematopoietic stem cells.

The proliferation rate of murine hematopoietic cells in regenerating spleen was assayed by a novel technique that analyzes the integrity of stem cells irrespective of their number. It relies on the increment of incorporation of 5-(125I) iodo-2'-deoxyuridine (125IUdR) at day 3 and 5 after transfusion of syngeneic marrow cells in fatally irradiated recipients. A prerequisite of the assay is the linearity between in fatally irradiated recipients. A prerequisite of the assay in the linearity between 125IUdR incorporation in the spleen and the number of cells transfused at both days of observation. The average increment of activity of 125IUdR from day 3 to 5 for the various graft size permits the description of a proliferation factor and from that a doubling time of the proliferating population in the spleen. Whole-body gamma-irradiation of donor mice causes a significant increase of doubling time in the grafts which persisted in part for 7 months after recovery from 500 rad exposure. The prolongation of doubling time expresses a residual injury and may interfere with the regulation of proliferation.

Residual damage and discontinuity of recovery in the hematopoietic system of mice following gamma-irradiation.

Recovery of the hematopoietic system up to 35 days following whole body irradiation of mice was investigated by measuring the repopulation ability of femoral bone marrow in irradiated recipients. The assay was performed five days after transfusion by testing for incorporation of 125iodo-deoxyuridine (125IUdR) into DNA of femoral marrow and spleen. The DNA incorporating cells are progeny of transfused stem cells or early precursors. For non-irradiated donors 125IUdR incorporation in the recipient spleens was proportional to the number of transfused cells between 0.05-1.0 x 10(6)/mouse but decreased with larger grafts (5-30 x 10(6). Yet, in the femur 125IUdR incorporation remained proportional over the range of 0.5-30 x 10(6) transfused cells. Recovery was discontinuous and not complete 35 days after irradiation. A possible explanation is injury to stem cells that still permits these cells to produce progeny within a milieu of high proliferation stimulus.

Cerebral glucose transport implies individualized glial cell function.

Previous positron emission tomography (PET) measurements of cerebral glucose transport using [11C]-3-O-methylglucose (CMG) suggested an interindividual variation in the values of the rate constant of tracer outflow (k2) larger than that for the clearance rate of inflow (K1). These two parameters were examined in healthy cerebral cortex by dynamic PET in 4 men and 2 women (aged 24 to 73 years) without neurologic disease, and in 1 man (42 years) with a recent left hemispheric cerebral infarction under normoglycemia (average blood plasma d-glucose concentration, 5.44 +/- 1.94 micromol/mL) and again under hyperglycemia (average, 10.24 +/- 1.44 micromol/mL). Time-radioactivity curves were obtained from healthy cortex (grey matter) and plasma and analyzed for the values of K1 and k2 by two graphical approaches and two fitting procedures. Both K1 and k2 significantly declined with increasing plasma glucose levels. A highly significant interindividual but not intraindividual variability for k2 was found at normoglycemia and hyperglycemia. The interindividual variability of K1, although borderline significant, was less than that of k2. Accordingly variable were the distribution volumes K1/k2. These data suggest individualized glial cell function and may be relevant to pathogenesis of neuropsychiatric disease.

Tomographic studies of rCBF with [99mTc]-HM-PAO SPECT in patients with brain tumors: comparison with C15O2 continuous inhalation technique and PET.

In 10 patients with malignant gliomas, the intracerebral distribution of [99mTc]-hexamethylpropylene-amine oxime [( 99mTc]-HM-PAO) was studied with single photon emission computed tomography (SPECT) in comparison with C15O2 steady-state inhalation technique to measure cerebral blood flow using positron emission tomography (PET). In all instances, the cerebral [99mTc]-HM-PAO distribution was comparable with the regional pattern of cerebral blood flow (rCBF) observed with PET. This was confirmed by a significant correlation of tumor to cortex and tumor to white matter ratios between these two experimental methods. However, the contrast between high and low activity regions in the SPECT scans was significantly less than that in the PET scans. Contrast enhancement of the SPECT scans was accomplished using a correction formula proposed by Lassen.

Pharmacokinetics of thallium-201 in normal individuals after routine myocardial scintigraphy.

Data of pharmacokinetic distribution of and radiation dose from 201Tl chloride used in routine myocardial scintigraphy are based on animal studies or on small groups of humans not exercised. In order to obtain data under routine conditions pharmacokinetics of 201Tl were measured in 15 individuals who had undergone diagnostic myocardial scintigraphy and were classified as normal. Ventral and dorsal whole-body scans were acquired until 9 days after injection. Conjugate pixels were averaged geometrically. Percentage values of total administered dose were obtained for total body and 13 organs by using a calculation method that takes into account the differentiation of overlapping organs.

In vivo assessment of phagocytic properties of Kupffer cells.

Three-compartment analysis was used to assess the kinetics of phagocytosis of Tc-99m-labeled human serum albumin microparticles (Tc-99m HSA-MM) in human Kupffer cells in vivo. The tracer turnover in these phagocytic cells could be described by a monoexponential accumulation with a two-stage elimination phase. Three-compartment analysis of the Tc-99m HSA-MM kinetics allowed us to quantify tracer attachment, phagocytosis, and degradation in Kupffer cells. The calculated time course of phagocytosis in ten control subjects proved to be identical to that of phagocytosis of various test substances in mouse macrophage monolayers (1). In addition, an impairment of particle turnover at the macrophage membrane, a significantly diminished (p less than 0.01) phagocytosis rate of the tracer, was observed in ten patients with various tumors.

Glucose transport and utilization in the human brain: model using carbon-11 methylglucose and positron emission tomography.

3-0-[11C]-Methyl-D-glucose (CMG) is specifically suited for measuring carrier facilitated glucose (G) transport; it enters the free G pool in tissue from where it is not utilized for metabolism in contrast to G, but is transported back into circulation. The ratio of carrier affinity for G and CMG was reported to be 1.11. By simultaneously measuring CMG concentration in plasma and in cerebral cortex in vivo with positron tomography at 1-min intervals for 40 min, two time-activity curves are obtained, as reported previously, which together with the G concentration in plasma yield the in vivo rate constants of G transport across the blood-brain barrier and the rate of G inflow; a repeat measurement at a different G concentration in plasma gives the in vivo Michaelis-Menten constant KM and the maximal rate of transport VMAX. The present paper summarizes and extends this approach to analyzing the free G pool in tissue, the rate of G return to circulation, and the rate of G exit into metabolism with its corresponding rate constants. The data from six volunteers agreed with results reported for the individual biochemical parameters in primate brains.

Measurement of pharmacokinetics of yttrium-86 radiopharmaceuticals with PET and radiation dose calculation of analogous yttrium-90 radiotherapeutics.

This study was performed to demonstrate the quantitative in vivo assessment of human pharmacokinetics of 90Y-radiotherapeutics using the positron-emitting substitute 86Y and PET. This technique is illustrated in a patient with disseminated bone metastases from breast cancer who was injected with 100 MBq of 86Y-citrate as an analog of the commercially available radiotherapeutic 90Y-citrate. Whole-body distribution was measured with a PET camera 4, 10, 21, 28 and 45 hr postinjection. Uptake data were determined from reconstructed transverse PET images by regions of interest placed in normal bone tissue, liver and metastases. Images of coronal and sagittal whole-body sections were obtained by reformatting the transverse PET images. The ratio of activity concentration in metastases to that in normal bone ranged from 1.5:1 to 3.5:1. Of the injected tracer, 13.4% was found in the skeleton and 0.43% in the metastasis with the highest 86Y concentration. Radiation doses per 1 MBq of injected 90Y-citrate were calculated from 86Y-citrate data and data from MIRD pamphlets 5 and 11. The doses were 1.01 MGy/MBq for red marrow, 593 microGy/MBq for the liver and approximately 3.5 MGy/MBq for the most conspicuous metastases. This study demonstrates that the use of PET via 86Y allows an individual in vivo quantification of activity uptake and radiation dose of both normal tissue and tumor in pain treatment with 90Y-labeled radiotherapeutics.

Lipid metabolism in the liver studied in vivo with two isomers of labeled fatty acid analogs.

UNLABELLED: The two radioiodinated fatty acid analogs 15-(para-131 I-phenylpentadecanoic acid (pPPA) and 15-(ortho-131I-phenyl)-pentadecanoic acid (oPPA) are isomers with individually different routes in lipid metabolism but with near equal transport kinetics into tissue. METHODS: Normal adult male Wistar rats (n = 79) and those with liver cell damage from adriamycin treatment (n = 84) received 1.48- 1.85 MBq 131I-pPPA or 131I-oPPA (specific activity, 33.3-46.3 GBq/microM) into the jugular vein. At 1, 2, 3, 5, 7, 10 and 20 min, livers of up to five animals per group were examined for total tracer uptake and tracer incorporation into various lipid fractions. RESULTS: Uptake of both isomers into the total liver plateaued at about 2 min; the ratio oPPA/pPPA in normal liver averaged 2.63 and was significantly higher than the average ratio of 1.50 after adriamycin treatment. This fall in ratio was mainly due to an increase of pPPA uptake. Significant differences of the respective ratios were found in the plateau for the phospholipids (9.7 versus 3.0), cholesterol (2.4 versus 0.7) and triglycerides (2.0 versus 0.4). CONCLUSION: The dual-tracer technique with pPPA and oPPA promises to be clinically useful for the diagnosis of liver disease by imaging the ratios of tracer uptake in the total liver and by in vitro analysis of the uptake ratio in serum triglycerides.