Factors Influencing the Uptake of Iron and Plutonium into Cells

Summary

Uptake of ⁵⁹Fe as well as ¹²⁵I-labelled Fe-transferrin into HeLa cells points to the existence of a limited number of specific binding sites. This is in contrast to hepatocytes and hepatoma cells (Hep G2) where metal uptake from transferrin is very low, not saturable and cannot be prevented by an excess of the protein. Iron uptake into these cells is much higher from the citrate complex. The same is true for plutonium uptake into rat hepatocytes, while the uptake of this metal into Hep G2 cells is very small regardless of the ligand. In contrast to iron, plutonium presented as citrate is taken up into HeLa cells much better than plutonium presented as transferrin.

The uptake of both metals from the citrate complex requires a high activation energy and can be prevented only by inhibition of oxidative phosphorylation. Other processes such as endocytosis, intactness of microtubuli, assembly of microfilaments or pH of the lysosomes do not seem to be of importance.

Metal uptake from the citrate complex can be prevented only by the presence of other chelating agents and/or by transferrin. It can be assumed, therefore, that the metals react directly with constituents of the cell membrane, a process in which chelating agents can successfully compete if they form strong enough complexes with the metals.     

Uptake and Clearance of Plutonium-238 from Liver Cells Transplanted into Fat Pads of F344 Rats

Summary

This research is directed toward understanding the role of liver cells and the liver environment in plutonium biokinetics. Animals injected with liver cells and control animals received a single intraperitoneal injection of 37 kBq (1 µCi) ²³⁸Pu citrate and were serially sacrificed 1, 5, 10, 15, 30, 60 or 70 days later. Uptake, retention and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. From these measurements, it was observed that the cells of the intact liver took up about twice as much ²³⁸Pu as liver cells transplanted into the fat pads of the same animal. The retention half-life was 8·3 days for the total activity in the liver, 20 days using tracks/cell measurements in the liver and 16 days for the tracks/cell measurements in the liver cells translocated to fat pads. When the data on tracks/cell were standardized relative to the amount of Pu present at 5 days after the injection, there was no significant difference between the retention of Pu in liver cells from intact animals and liver cells transplanted into the fat pads. About 20 per cent of the 5-day Pu liver burden in both liver cells and liver cells transplanted into fat pads was retained at 70 days. The smaller retention and clearance for liver cells in different environments indicate that uptake and clearance of Pu from the body is dependent, to a major extent, upon hepatocyte function.     

Comparative tissue uptake and cellular deposition of three different plutonium chemical forms in rats

Purpose: To evaluate the influence of the chemical form of plutonium (Pu) on its distribution in tissues and within liver cells populations.

Materials and methods: Groups of male Sprague–Dawley rats were contaminated by intravenous injection of either Pu citrate, Pu nitrate or Pu phytate. Pu content was determined in various tissues at different times after injection. Pu liver distribution was analysed by autoradiography and after cellular separation.

Results: Biokinetic studies indicate that Pu citrate and Pu nitrate predominantly retained in the skeleton within the first hours after injection, whereas most of the Pu was in the liver after injection of Pu phytate. Autoradiographs showed that Pu citrate was homogeneously distributed in the liver, while Pu nitrate accumulated into ‘hot points’. Pu phytate showed an intermediate distribution pattern. Hepatic cell separation revealed a difference of uptake between the two cell types depending on the chemical form of injected Pu, and on the time after contamination.

Conclusions: Distinct Pu behaviour was observed for biokinetics, retention and liver distribution. The large differences noted between citrate, nitrate and phytate might be explained by differences in systemic and hepatic transport.     

Long-term Effects of Plutonium-239 and Radium-224 on the Distribution and Performance of Pluripotent Haemopoietic Progenitor Cells and Their Regulatory Microenvironment

Summary

Experiments are described which investigate the long-term damage to haemopoietic progenitor cells (CFU-S) and their microenvironment in mouse marrow resulting from the administration of leukaemogenic amounts of plutonium-239 and radium-224. ²³⁹Pu (35 Bq g^?1 body weight) and ²²⁴Ra (555 Bg g^?1 body weight) were injected into 10–12-week-old mice, and numbers, proliferative activity and self-renewal capacity of CFU-S were measured at different locations in femoral marrow at intervals over the following 2 years. Parallel measurements were also made of the quality of the haemopoietic microenvironment by ectopic transplantation of bone marrow cells. There was some recovery from the initial effects of ²³⁹Pu on CFU-S numbers after 3–6 months, although the recovery was not maintained in all marrow fractions. Following ²²⁴Ra administration there was an initial transient increase in CFU-S numbers in the fraction of marrow furthest from bone surfaces but a considerable depression in numbers in other regions of marrow; there was no recovery between 3 and 6 months and subsequent recovery was not complete in all regions of marrow. The differential responses of CFU-S and the haemopoietic microenvironment following ²²⁴Ra or ²³⁹Pu administration seemed in some ways related to the metabolism of the radionuclides. There was a profound reduction in the ability of marrow to generate ossicles when transplanted under the kidney capsule as a result of the administration of either ²²⁴Ra or ²³⁹Pu, with only transient recoveries from the effects of ²³⁹Pu at 4 days and at 3 months after injection.     

Relation between 67Ga uptake and iron metabolism in rat tissues

The relationship between ⁶⁷Ga uptake and iron metabolism was investigated in rat tissues. ⁶⁷Ga and ⁵⁹Fe(II) both accumulated in the mitochondrial-lysosomal fraction after being injected. Moreover, they both showed especially high affinity for heparan sulfate (HS) among various acid mucopolysaccharides (AMPS). When iron (ferrous citrate) was injected IV before, simultaneously with, and after ⁶⁷Ga citrate IV injection, ⁶⁷Ga uptake was significantly inhibited in normal rat liver in all cases. Elevated ⁶⁷Ga uptake in the liver of CCl₄-treated rats was also lowered to the control level by iron pretreatment. High zinc intake remarkably elevated the ⁶⁷Ga uptake in rat liver. The contents of iron in the liver and liver AMPS of 0.75% zinc-fed rats were lowered in comparison with those in controls. Thus, the elevation of ⁶⁷Ga uptake in the liver of zinc-fed rats might be due to the decreased of iron bound to HS.     

Biochemical Binding and Distribution of Protactinium-233 in the Rat

Summary

Following intravenous injection into male Sprague–Dawley rats ²³³Pa, like other elements, deposits predominantly in the skeleton (ca. 70–80 per cent), but unlike Pu and Am the liver deposition of ²³³Pa is low, about 2–3 per cent between 1 and 7 days. About 99 per cent of the injected ²³³Pa is lost from the plasma compartment in 3 days, a clearance comparable to that of Pu but much slower than that of Np, Am or Cm. On entering the liver cell cytosol ²³³Pa is bound rapidly to an unidentified protein of molecular mass 200 kDa and to a protein of 80 kDa, which is probably transferrin. Within a few hours the metal migrates to bind to a protein of > 400 kDa which has been tentatively identified as ferritin. Some ²³³Pa remains bound to small ligands until virtually all the intracellular ²³³Pa has been deposited in the lysosomes, or to a lesser extent in some other, as yet, unidentified organelles.     

In vitro uptake of gallium and chlorpromazine by mouse tumour cells

Primary cell suspensions were prepared from mouse sarcoma by enzymatic digestion with pronase. The cells were incubated with gallium citrate Ga 67 or the basic drug ¹⁴C-chlorpromazine (CPZ) for up to 1 h at 37° C, and the label uptake was determined. The Ga uptake was proportional to time (0–30 min), whilst the CPZ uptake rapidly reached apparent saturation, (10 min). Metabolic inhibitors did not affect label uptake; however, disrupting the cell membrane with n-ethylmaleimide or heating at 56° C significantly reduced CPZ accumulation but did not inhibit Ga uptake. Ga accumulation was decreased by adding human transferrin (0.5 mg/ml). Both gallium and chlorpromazine are fixed in the lysosomes of cells; however, in this system, they appeared to enter mouse sarcoma cells by different energy-independent mechanisms. The Ga uptake may reflect adsorption to cell components, whilst CPZ uptake required an intact cell and may be due to passive diffusion.     

DNA Strand Breaks from Tritium Decay: A Local Effect for Cytosine-6-3H

Summary

Twelve-week-old female (C3H × 101)F₁ mice were injected intravenously with an ultrafiltered solution of ²³⁹Pu in 1 per cent trisodium citrate, and mated to uninjected PCT males. The plutonium content was examined radiochemically and autoradiographically in placentae and foetuses on the 12th and 18th days of gestation, and in neonates during the 24 hours after birth and also at 18 days post-natally.

Plutonium was distributed in most tissues of the late foetus and the suckling as it is in adult mice. However, on both the 12th and 18th days of gestation the concentration in the yolk-sac splanchnopleure was much higher than in any other foetal tissue. The amount of ²³⁹Pu in 18-day-old sucklings was between two and seven times as great as in 1-day-old neonates because of ingestion of milk from the lactating dams. In the first litter following administration of the radionuclide to the dam, about 0·02 per cent of the plutonium injected was transferred to an individual offspring by the time of birth, and a further 0·08 per cent by the time of weaning.     

Comparative tissue uptake and cellular deposition of three different plutonium chemical forms in rats

PURPOSE: To evaluate the influence of the chemical form of plutonium (Pu) on its distribution in tissues and within liver cells populations. MATERIALS AND METHODS: Groups of male Sprague-Dawley rats were contaminated by intravenous injection of either Pu citrate, Pu nitrate or Pu phytate. Pu content was determined in various tissues at different times after injection. Pu liver distribution was analysed by autoradiography and after cellular separation. RESULTS: Biokinetic studies indicate that Pu citrate and Pu nitrate predominantly retained in the skeleton within the first hours after injection, whereas most of the Pu was in the liver after injection of Pu phytate. Autoradiographs showed that Pu citrate was homogeneously distributed in the liver, while Pu nitrate accumulated into 'hot points'. Pu phytate showed an intermediate distribution pattern. Hepatic cell separation revealed a difference of uptake between the two cell types depending on the chemical form of injected Pu, and on the time after contamination. CONCLUSIONS: Distinct Pu behaviour was observed for biokinetics, retention and liver distribution. The large differences noted between citrate, nitrate and phytate might be explained by differences in systemic and hepatic transport.     

In vitro binding of 67Ga to isolated rat liver cells

In order to understand the mechanism of the accumulation of gallium citrate (Ga-67) in normal liver, an in vitro investigation system was developed using isolated rat liver cells, and various basal factors relating to ⁶⁷Ga binding to normal rat liver cells were studied. In this study, the values of ⁶⁷Ga binding to the liver cells increased in parallel with the number of cells; however, binding was hardly affected by higher doses of ⁶⁷Ga. The binding of ⁶⁷Ga to the cells was inhibited by the presence of ethylenediaminetetraacetate (EDTA) and citrate. Phosphate and bicarbonate (10^-2 M) slightly inhibited ⁶⁷Ga binding to the cells. The binding of ⁶⁷Ga to the cells increased as the pH was decreased. These results suggest that ⁶⁷Ga binding to normal rat liver cells may occur in free gallium. Moreover, the utilization of isolated rat liver cells is useful for understanding the ⁶⁷Ga accumulation mechanism in normal liver.     

In vitro comparison of renal handling and uptake of two somatostatin receptor-specific peptides labeled with indium-111

Objective  Radiolabeled receptor-specific somatostatin analogs labeled with gamma- or beta-emitting radionuclides are useful for scintigraphic imaging and/or therapy of selected neuroendocrine tumors. However, significant renal uptake may result in radiotoxicological injury of the kidney and can limit clinical application of the agents. The aim of the study was to analyze renal handling, rate, and mechanism of renal accumulation of two somatostatin receptor-targeted peptides, [DOTA⁰, Tyr³, Thr⁸]-octreotide (DOTA-TATE) and [DOTA⁰, 1-Nal³]-octreotide (DOTA-NOC), labeled with indium-111 using in vitro methods. Methods  The perfused rat kidney and freshly isolated rat renal cells were used as experimental models. The perfusion was performed in a recirculation regimen at constant pressure with solution containing bovine albumin, erythrocytes, and a mixture of essential substrates. The renal cells were isolated from rat kidneys using two-phase collagenase perfusion. Accumulation studies were used to evaluate the renal uptake of the peptides and to compare their accumulation with that of passively or actively transported model drugs. The influence of selected inhibitors of receptor-mediated endocytosis and the inhibition of energy-dependent transport processes on the uptake were also investigated using isolated renal cells. Results  The renal clearance of ¹¹¹In-DOTA-NOC in the perfused rat kidney was significantly lower than that of ¹¹¹In-DOTA-TATE. Reverse situation was found in the case of renal retention. Pretreatment of the perfused kidney with maleate markedly decreased the renal retention. ¹¹¹In-DOTA-NOC was accumulated in the isolated renal cells at a higher rate than ¹¹¹In-DOTA-TATE (ratio 3: 1). The uptake of the radiopeptides in renal cells was higher than the uptake of not only the passively transported sucrose but also actively transported and accumulated methylglucose. The rank order of potency to inhibit the uptake by active endocytosis was approximately aprotinin > maleate > lysine. The uptake of the radiopeptides in the renal cells was temperature dependent. Conclusions  Both in vitro methods showed a higher renal accumulation of ¹¹¹In-DOTA-NOC in comparison with ¹¹¹In-DOTA-TATE. The renal uptake was partly decreased by inhibitors of receptor-mediated endocytosis and by a block of energy-dependent processes. A significant participation of active transport processes in renal accumulation of the studied peptides was confirmed.     

Efficacy and Toxicity of NN′N″N‴-tetra(2,3,4-trihydroxybenzoyl)-spermine for Decorporation of 239Pu from Mice

Summary

Male SAS/4 mice were injected i.v. with 6·6 kBq ²³⁹Pu-citrate. After 1 or 24 h a single i.p. injection of 15 or 30 μmol kg^?1 or repeated (three or four) daily injections of 30 μmol kg^?1 of tetra-THB-spermine were given, and at 4 or 7 days Pu retention was measured in liver, kidneys and femur. Besides tetra-THB-spermine, equimolar doses of tetra-DHB-spermine were injected for comparison, or equimolar doses of diethylene triamine-pentaacetic acid (DPTA) as a reference compound. Histological changes in kidneys and liver were examined after i.p. injections of 30 μmol kg^?1 or at 2–13 times higher doses of tetra-THB-spermine. The results show that: (1) Introduction of an additional hydroxy group into the aromatic moieties of tetra-DHB-spermine results in increased hydrophilicity, lower toxicity and a lower renal retention of Pu. (2) Tetra-THB-spermine and tetra-DHB-spermine are similarly effective in removing plutonium from liver and bone. Their efficacies in removing Pu from bone are approximately similar to those of DTPA but for whole-body removal they are generally inferior. (3) Multiple (30 μmol kg^?1) of tetra-THB-spermine were no more effective than a single injection at mobilizing Pu from the liver. (4) Four injections of tetra-THB-spermine induced cloudy swelling and fatty degeneration in epithelial cells of the proximal convoluted tubules. At levels of 400 μmol kg^?1 tetra-THB-spermine produced severe degenerative glomerular lesions, foci of liver necrosis and thromboses of the portal vein branch.     

Remeasurement of 241Pu half life by gamma-ray spectrometry

The half life of ²⁴¹Pu has been remeasured to be 14.355 y with an estimated standard deviation of 0.040 y (a mean tropical year of 365.242 d) by a new method based on high-resolution γ-ray spectrometry. During 10 y, 156 sets of normalized spectral full-energy peak-area ratios (proportional to ²⁴¹Pu/²³⁹Pu mass ratios) from 13 plutonium samples were obtained by γ-ray spectrometry. The ²⁴¹Pu half-life value was extracted by an appropriate statistical analysis of the measured ratios. In the method used, the measured ratios are essentially independent of plutonium sample properties, of counting geometry, and of detector efficiency as well as of pile-up and deadtime. The much longer-lived ²³⁹Pu served as a monitor by which differences in detection efficiency between ²⁴¹Pu/²³⁹Pu γ-ray pairs were normalized, and by which implicit corrections for electronic counting losses were made.     

Dynamic evaluation of the hepatic uptake and clearance of manganese-based MRI contrast agents: A 31P NMR study on the isolated and perfused rat liver

This spectroscopic study compares the mechanisms of the hepatic uptake of manganese chloride (MnCl₂) and manganese dipyridoxyl diphosphate (MnDPDP). Alterations of the phosphorus-31 (³¹P)-NMR spectrum of the intracellular adenosine 5′-triphosphate (ATP) are used to monitor the internalization of manganese by the isolated and perfused rat liver. Mn²⁺ delivered as MnCl₂ in the perfusate rapidly enters the hepatocytes, where it strongly interacts with ATP, inducing a broadening of the ³¹P lines. The inhibition of the process by nifedipine confirms that manganese ions cross the cellular membrane at least partly through Ca²⁺ channels. MnDPDP induces weaker but still significant changes of the ATP spectrum. The inability of pyridoxine to compete for the uptake of manganese confirms that the vitamin B₆ carrier is not involved in the internalization process of the paramagnetic complex. Finally, preincubation of MnDPDP with blood does not increase the extent of the dissociation. The alterations of the ³¹P spectrum of the liver subsequent to the administration of MnDPDP are attributable to a fraction of free Mn²⁺ released by the chelate and delivered to the hepatocytes.     

Hypoxia-selective Radiosensitization of Mammalian Cells by Nitracrine,an Electron-affinic DNA Intercalator

Summary

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4°C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1·6 at 1 µmol dm^?3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4°C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH 7 of ?275mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.     

31P NMR measurements of the effects of unsaturated fatty acids on cellular phospholipid metabolism

³¹P NMR measurements on extracts prepared from a variety of cultured mammalian cell lines and primary rat hepatocytes have shown changes in the levels of several phospholipid metabolites after incubation of cells with unsaturated fatty acids. These data suggest a possible link between the accumulation of neutral lipid and the changes in phospholipid metabolite concentrations that have been observed in some tumor cells and other rapidly growing tissues such as the regenerating liver and mitogen-stimulated lymphocytes.     

The relationship between Ga-67 accumulation and cell cycle in malignant tumor cells in vitro

Previously we reported that the deposition of ⁶⁷Ga into malignant tumor may be a sensitive index of proliferative activity in tumor cells. For the purpose of elucidation of this hypothesis, we investigated the relationship between the accumulation of ⁶⁷Ga into malignant tumor cells and the cell cycle in vitro. We discovered that the uptake of both ⁶⁷Ga and ⁵⁹Fe into synchronized mouse tumor cells reaches a peak at the G2 stage which precedes cellular proliferation. Both iron and transferrin are specifically required by cells in culture for cell division, and the fact that ⁶⁷Ga and ⁵⁹Fe uptake into tumor cells peaks at the G2 stage of the cell cycle suggests that there is a correlation between ⁶⁷Ga uptake and the rate of cellular proliferation in malignant tumor cells.     

Studies of the myocardial uptake and excretion mechanisms of a novel 99mTc heart perfusion agent

Introduction^99mTc-TMEOP is a novel heart perfusion radiotracer exhibiting high initial and persistent heart uptake associated with rapid blood and liver clearance. This study aimed at determining the mechanisms of myocardial localization and fast liver clearance of ^99mTc-TMEOP.MethodsSubcellular distribution of ^99mTc-TMEOP was determined in excised rat heart tissue by differential centrifugation. The effect of cyclosporin A on the pharmacokinetic behaviour of ^99mTc-TMEOP was evaluated by both ex vivo biodistribution and in vivo planar imaging studies.ResultsSubcellular distribution studies showed that more than 73% of ^99mTc-TMEOP was associated with the mitochondrial fraction. Comparison with subcellular distribution of ^99mTc-sestamibi showed no significant difference in the mitochondrial accumulation between the two tracers. Biodistribution studies in the presence of cyclosporin A revealed an increase in kidneys and liver uptake of ^99mTc-TMEOP, suggesting the involvement of multidrug resistance transporters in determining its pharmacokinetic profile.ConclusionsThe heart uptake mechanism of ^99mTc-TMEOP is similar to that of the other reported monocationic ^99mTc cardiac agents and is associated with its accumulation in the mitochondria. Cyclosporin A studies indicate that the fast liver and kidney clearance kinetics is mediated by P-glycoprotein (Pgp), supporting the potential interest of this radiotracer for imaging Pgp function associated with multidrug-resistant tumours.     

Enhancement of hepatic gallium-67 uptake by asialo-transferrin

A significant fraction of intravenously injected 67Ga localizes in the liver. The mechanism of 67Ga uptake by the liver is not clear. Hepatocyte membranes contain galactose-specific receptors which selectively remove asialo-glycoproteins from the circulation by way of endocytosis. In this investigation, we examined whether endocytic uptake of asialo-transferrin would involve 67Ga transport into hepatocytes. We demonstrated that asialo-transferrin bound 67Ga as effectively as apotransferrin. Human asialo-transferrin markedly enhanced 67Ga uptake by isolated, perfused rat livers. Human asialo-orosomucoid, but not orosomucoid competitively inhibited hepatic uptake of the 67Ga asialo-transferrin complex, indicating that hepatic 67Ga uptake in the presence of asialo-transferrin occurred by way of galactose-specific receptors. Our results point to a novel pathway for metal ion transport into hepatocytes by way of galactose receptor mediated endocytosis.