The induction of liver tumors by 239Pu citrate or 239PuO2 particles in the Chinese hamster

The influence of radiation dose distribution on the frequency of 239Pu-induced liver tumors was evaluated in the Chinese hamster. Different concentrations of 239Pu citrate 239PuO2 particles of known sizes were injected intravenously via the jugular vein. About 60% of the injected 239Pu citrate was deposited in the liver and 40% in the bone. The 239Pu citrate was rather uniformly distributed throughout the liver parenchyma. Injected plutonium oxide particles were taken up by the reticuloendothelial system with 90% of the body burden deposited in the liver. The 239PuO2 particles were localized in the Kupffer cells and produced nonuniform dose distributions that were dependent on particle size. There was an activity- and dose-dependent increase in the incidence of total liver parenchymal cell tumors following injection with either plutonium particles or citrate. For animals that received 14.0-, 2.7-, 0.3-, and 0.04-Gy dose to liver from 239Pu citrate the cumulative tumor incidence was 39, 32, 5, and 0%, respectively. Animals that were injected with the 0.24 micron 239PuO2 particles had doses of 42.0, 7.2, and 0.8 Gy to the liver and tumor incidences of 34, 26, and 5%, respectively. Plutonium citrate also produced hemangiosarcomas of the liver and tumors in bone and bone marrow. The latent period for liver tumor appearance in animals exposed to 239Pu citrate or 239PuO2 particles increased as the injected activity decreased. For animals injected with a similar total activity (7.4 Bq/g), the lifetime cumulative liver tumor incidence was similar for animals exposed to either 239Pu citrate (32%) or 239PuO2 (26%). There was little effect of particle size on liver tumor incidence. These data indicate that, in Chinese hamster liver, local radiation dose distribution is less important in altering tumor incidence than injected activity or average dose. However, the more uniform irradiation from 239Pu citrate administration was more effective in cancer production than the nonuniform irradiation from 239PuO2 particles.     

Role of transferrin in uptake of non-physiological metals into cells

At physiological concentrations of citrate the uptake of 59Fe, 67Ga, and 239Pu into human type B lymphocytes of splenic origin is the same in viable and in non-viable cells. Addition of transferrin has no effect on the uptake into non-viable cells but in viable cells it increases the uptake of Fe and Ga but decreases that of Pu. Uptake decreases as transferrin concentration increases although this is less marked with Ga.     

Alkaline phosphatase and transaminase activity in the liver and blood serum of rats in the late periods following combined exposure to external gamma radiation and alpha radiation from plutonium-239

A study was made of activity of alkaline phosphatase and alanine- and aspartate aminotransferase in rat liver and blood serum at remote times after external gamma-irradiation combined with internal exposure to 239Pu nitrate delivered in two chronically effective doses. The radionuclide was shown to be mainly responsible for the changes observed in activity of the enzymes under study. The degree to which the changes were manifest depended upon dose of plutonium administered.     

The ultrastructure of mouse testicular interstitial tissue containing plutonium-239 and its significance in explaining the observed distribution of plutonium in the testis.

The technique of autoradiography with Araldite-embedded sections was used to study the distribution of 239Pu in mouse testis at various times post-injection. Adjacent sections were examined with both the light microscope and electron microscope. The autoradiographs showed that from 1 week to 3 months postinjection, most 239Pu in located in interstitial tissue. The major change in distribution observed was that the early diffuse deposit in interstitial tissue is concentrated in macrophages with increasing time post-injection. This is a real change of distribution as the amount of 239Pu in mouse testis remains constant from 1 week to 3 months post-injection. Study of the ultrastructure of interstitial tissue indicated that the accumulation of 239Pu in macrophages may be brought about in two ways. First, there may be phagocytosis of dead cells containing 239Pu. Second, 239Pu may follow the transfer of waste products of hormone synthesis from Leydig cells into macrophages. The significance of these observations is discussed with regard to the deposition of 239Pu in human testis.     

Role of transferrin in metal uptake by human lymphoblasts in vitro

The uptake and binding of 59Fe, 67Ga and 239Pu complexed with citrate of transferrin (Tf) and of 125I-labelled Fe-Tf by human lymphoblasts (WI-L2 cells) have been studied. Uptake kinetics of 59Fe-Tf and [125I]-Tf point to internalization by receptor mediated endocytosis. 67Ga binding and uptake is always less. This may be explained by a lower affinity of Ga-complexes for the cell surface. Factors which influence Fe uptake have a similar effect on Ga. 239Pu uptake and binding, however, are different, especially in that Tf does not stimulate 239Pu uptake and may actually decrease it.     

Comparative studies on the lysosomal association of monomeric 239Pu and 241Am in rat and Chinese hamster liver: analysis with sucrose, metrizamide, and Percoll density gradients of subcellular binding as dependent on time

The binding of 239Pu and 241Am in the livers of Chinese hamsters and rats was analyzed by centrifugation of a mitochondrial-lysosomal fraction in sucrose, metrizamide, and Percoll density gradients at intervals between 4 and 70 days after nuclide injection. The behavior of 239Pu and 241Am during the centrifugation experiments was very similar. In contrast to the results for rats, the median densities of the nuclide profiles from hamsters decrease with time in hyperosmolar sucrose gradients, as does the nuclide fraction liberated by addition of Triton X-100, and the nuclide profiles do not respond typically to Triton WR 1339 treatment of the animals. The results with nearly iso- osmolar metrizamide gradients, together with those for Percoll, agree with the assumption that there is an initial lysosomal association of the transuranium elements. It was concluded from the results that the major fraction of 239Pu and 241Am remained bound to typical lysosomes in rat liver, whereas those in hamster liver may be transformed into telolysosomes . Possibly, a vesicular biliary transport system for certain heavy metals, for which evidence exists in rat liver, does not occur in Chinese hamster liver.     

Distribution and biological effects of inhaled 239Pu(NO3)4 in cynomolgus monkeys.

Twenty male cynomolgus monkeys were exposed by inhalation either to an aerosol of 239Pu(NO3)4 to produce projected initial lung burdens of either 40, 10, or 4 kBq or to a carrier aerosol as a control. Animals died or were sacrificed at 0.01, 1, 3, 6, 12, 24, 40, and 99 months after inhalation, and the distribution and biological effects of the 239Pu were determined. The 239Pu cleared efficiently from the lungs so that less than 0.05 kBq remained at 99 months after exposure to 40 kBq. Total skeletal 239Pu activity was nearly constant after the first year, but the fraction of the body burden in skeleton at sacrifice increased with time up to 99 months because of clearance from other organs. Plutonium in the liver increased to a peak at 1 year and then decreased to about 10% of the peak value at 99 months. Plutonium in the testes was localized in the interstitial tissue with only 0.01 to 0.002% of the projected lung burden remaining in testes at 99 months after inhalation. Three animals exposed to 40 kBq of 239Pu died of radiation-related pulmonary pneumonitis and fibrosis. A primary papillary adenocarcinoma of the lung was identified in one animal exposed to 40 kBq initial lung burden and sacrificed 99 months after inhalation. The frequency of chromosome aberrations in blood lymphocytes was significantly elevated only in monkeys with projected deposits of 40 kBq of 239Pu. There was no change in aberration frequency in other exposure groups as a function of inhaled activity, time after exposure, or calculated total dose to the lungs. Only in monkeys that had marked radiation-induced pathological changes in the lung did the frequency of chromosome-type aberrations increase significantly, to a value about twice the control level. In cynomolgus monkeys, chromosome aberration frequency in blood lymphocytes is not a good indicator of radiation dose or damage from inhaled soluble plutonium.     

Removal of monomeric 239Pu from bones and liver by liposome-encapsulated pentacin

Dependence of monomeric 239Pu removal from the liver and skeleton by liposome-encapsulated pentacine on dose and concentration of encapsulated chelate was studied in rats. It has been shown that the liposome-encapsulated pentacine (LP) removed 1.5-2.5 times as much 239Pu as free chelate (FP). Dose-effect dependences were logarithmic. The distinction between LP and FP in 239Pu removal from the liver was maximum when chelate had been used in a dose of 50 mumol/kg, with the dose effect upon injection in a large number of liposomes (200 mumol of lipids/kg) being 1.8 times as high as upon injection in smaller number of liposomes (50 mumol/kg). LP doses varying from 100 to 400 mumol/kg, there were no differences between two types of LP; with a LP dose of 400 mumol/kg its action is a bit stronger than that of the chelate. The distinction between LP and FP in 239Pu removal from the skeleton is the greatest with chelate doses exceeding 100 mumol/kg. The use of liposomes in combination with concentrated chelate solution is more effective. Possible interpretation of the features revealed are discussed.     

Biochemical binding and distribution of protactinium-233 in the rat

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 greater than 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.     

Efficacy of polymeric 239Pu removal by liposome-encapsulated pentacin

Liposome-incapsulated pentacine (DTPA) removes intracellular polimeric 239Pu and colloidal hydroxide of polymeric 239Pu from a rat body in the amounts 2- and 4 times, respectively, exceeding those eliminated by free DTPA. However the amount of 239Pu removed decreases sharply with increasing 239Pu hydrolysation and polymerization. It is concluded that the effectiveness of 239Pu removal depends on the physico-chemical status of the radionuclide and its interaction with the biosubstrate rather than on the amount of DTPA entering the cells.     

The subcellular distribution of 238Pu and 239Pu in primary cultures of rat hepatocytes

The subcellular distribution of 238Pu and 239Pu after incubation of primary cultures of rat hepatocytes with the citrate complex of these metals was studied, and the results were compared with data from in vivo experiments. As in vivo, the lysosomes are the principal organelles in which 238Pu and 239Pu are accumulated. In contrast to in vivo studies, 239Pu is also detectable on the pericellular membranes and in the cell nuclei, where it is predominantly bound to a high-molecular-weight component. The percentage of the total cellular 239Pu which can be recovered in the cell nuclei increased with incubation time from 10% at 1 h to nearly 30% at 5 h. Plutonium-238, an isotope with 270-fold higher specific activity than 239Pu, showed no association with the nuclei. The membrane-bound fraction of 239Pu, as determined using the exogenous chelator diethylenetriaminepentaacetic acid decreased from 30% at shorter incubation times to 15% at longer incubation periods. After incubation with 238Pu the membrane fraction and the cytosolic fraction contained higher concentrations of the radionuclide than after incubation with 239Pu.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

Iron metabolism pathways in the rat hepatocyte

A small to moderate inhibitory effect of iron uptake by isolated rat hepatocytes in short-term studies was seen with oxidative phosphorylation and electron transport inhibitors, and no inhibition by agents affecting pinocytosis. Intracellular transferrin was able to donate iron to the small-molecular weight iron pool, and the latter was able to transfer, by a process not requiring energy or movement of serum transferrin, iron to ferritin. Serum transferrin was not able to lose iron to any cytosol components. Reducing agents were not able to abstract iron from rat serum transferrin to any great extent. It is concluded that iron is taken up by the rat hepatocyte from serum transferrin by a process not requiring energy or movement of serum transferrin into the cell interior; and that intracellular transferrin is involved in acquiring iron from serum transferrin at the cell surface, with iron then being transferred to the small-molecular weight iron pool and hence to ferritin. It is also proposed that intracellular transferrins may have the general function of interacting with serum transferrin at cell surfaces.     

Purification and characterization of testicular transferrin secreted by rat Sertoli cells.

Sertoli cells synthesize and secrete a transferrin-like protein (testicular transferrin) [Skinner & Griswold (1980) J. Biol. Chem. 255, 1923-1925]. The purpose of the present study was to purify and characterize testicular transferrin and to compare it with serum transferrin. Testicular transferrin was obtained from the medium of cultured rat Sertoli cells, whereas serum transferrin was obtained from rat serum. Both proteins were purified with the use of phenyl-Sepharose hydrophobic chromatography and transferrin immunoaffinity chromatography. The purified proteins were shown to have similar molecular masses (75 000 Da) and amino acid compositions. The pattern of tryptic peptides from testicular and serum transferrin were found to be essentially the same when analysed by reverse-phase high-pressure liquid chromatography. The carbohydrate composition of both transferrins was determined by several colorimetric assays and g.l.c. Testicular transferrin, isolated from cell culture medium, had increased amounts of glucose, galactose and glucosamine. Serum transferrin that was incubated with cell culture medium also had a large amount of associated glucose. The results show that testicular transferrin and serum transferrin are structurally very similar and are possibly products of the same gene expressed in two different tissues, the testis and liver. However, the amount of carbohydrate associated with these two proteins is different.     

An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium

Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.     

Effect of reduced atmospheric pressure and fasting on transferrin synthesis in the rat

The concentrations of transferrin and albumin in the blood serum and microsomal fraction of the liver and the incorporation of [¹⁴C] leucine into the proteins were measured in rats which were fasted while exposed to ambient atmospheric pressure or to a pressure of one-half atmosphere. The rates of protein synthesis were estimated in a relative manner from the ratio of ¹⁴C incorporation into the two proteins and in an absolute manner using the liver free ¹⁴C and leucine concentrations to measure the specific activity of the precursor pool. Fasting at ambient pressure was accompanied by a decrease in the serum and microsomal concentrations of transferrin but not of albumin and by a marked decrease in the relative and absolute synthesis rates of transferrin. By contrast, fasting at reduced ambient pressure was associated with an increase in the serum transferrin concentration and in the relative and absolute rates of synthesis of the protein. It is concluded that fasting in the rat produces a much greater decrease in the rate of synthesis of transferrin than of albumin and that exposure to reduced ambient pressure stimulates transferrin synthesis but not albumin synthesis.     

Plutonium-239 deposition in the skeleton of the mouse.

Using the technique of neutron-induced autoradiography, together with computer-based methods of data reduction, the distribution of intravenously injected plutonium-239 in the skeleton of the female CBA mouse, 24 hours after injection, has been investigated. With these techniques, it is possible to measure the localization of 239Pu on the endosteal and periosteal surfaces of the bone to an accuracy of approximately +/- 2 x 5 micrometer. Results are reported for the distribution of 239Pu in the third lumbar vertebra, a central caudal vertebra, the right ilium and the right femur. Radiochemical analyses of the 239Pu in other comparable bones of the skeleton are also reported.     

Identification of transferrin as the main binding site for protactinium in rat blood serum

The distribution of ²³³Pa in rat serum at periods between 5 and 50 min after i.v. injection of a solution of protactinium chloride was studied by gel chromatography.Sequential analysis of sera on Sephacryl S-300 and DEAE-Sephadex showed that ²³³Pa was associated only with the transferrin fraction of the serum proteins. This finding was confirmed by iso-electric focusing electrophoresis. In the cytosol fractions prepared from the liver and kidneys of the ²³³Pa injected rats the nuclide was also shown to be protein bound.     

Uptake and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats

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 microCi) 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.