The assessment and management of risks associated with exposures to short-range Auger- and beta-emitting radionuclides. State of the art and proposals for lines of research.

The assessment and management of risks associated with exposures to ionising radiation are defined by the general radiological protection system, proposed by the International Commission on Radiological Protection (ICRP). This system is regarded by a large majority of users as a robust system although there are a number of dissenting voices, claiming that it is not suitable for estimating the risks resulting from internal exposures. One of the specific issues of internal exposure involves short-range radiations such as Auger and beta particles. Auger- and beta-emitting radionuclides can be distributed preferentially in certain tissue structures and even in certain cellular organelles, according to their chemical nature and the vector with which they are associated. Given the limited range of the low-energy electrons in biological matter, this heterogeneous distribution can generate highly localised energy depositions and exacerbate radiotoxic responses at cellular level. These particularities in energy distribution and cellular responses are not taken into account by the conventional methods for the assessment of risk.Alternative systems have been proposed, based on dosimetry conducted at the cellular or even molecular level, whose purpose is to determine the energy deposition occurring within the DNA molecule. However, calculation of absorbed doses at the molecular level is not sufficient to ensure a better assessment of the risks incurred. Favouring such a microdosimetric approach for the risk assessments would require a comprehensive knowledge of the biological targets of radiation, the dose-response relationships at the various levels of organisation, and the mechanisms leading from cellular energy deposition to the appearance of a health detriment. The required knowledge is not fully available today and it is not yet possible to link an intracellular energy deposition to a probability of occurrence of health effects or to use methods based on cellular dosimetry directly.The imperfections of the alternative approaches proposed so far should not discourage efforts. Protection against exposure to Auger and low-energy beta emitters would benefit from mechanistic studies, dedicated to the study of energy depositions of the radionuclides in various cellular structures, but also from radiotoxicological studies to define the relative biological effectiveness of the various Auger emitters used in medicine and of certain low-energy beta emitters, whose behaviour may depend greatly on their chemical form during intake. The scientific expertise, as well as the human and physical resources needed to conduct these studies, is available. They could be now mobilised into international low-dose research programmes, in order to ultimately improve the protection of people exposed to these specific radionuclides.

Detergent-solubilized proteoglycans in rat testicular Sertoli cells.

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.

IGF-1 stimulates synthesis of undersulfated proteoglycans and of hyaluronic acid by peritubular cells from immature rat testis.

The exposure of confluent peritubular (PT) cells from immature rat testis to insulin-like growth factor-1 (IGF-1) induced a time and dose-dependent increase of [35S]-sulfate and [3H]-D-glucosamine incorporations in newly synthesized proteoglycans (PG). This increased content of PG was the result of an enhancement of PG synthesis rather than a decreased rate of degradation. IGF-1 had no effect on the molecular weight of synthesized PG nor on the nature and distribution of the constitutive glycosaminoglycan chains, both in medium and in cell layer. The stimulation of PG synthesis by IGF-1 appeared to be due, at least partially, to an increase of glycosylation processes. IGF-1 effect was mediated by the classical tyrosine kinase signalling process, since IGF-1 action on PG synthesis was abolished by genistein and tyrphostin A9, two well known tyrosine kinase inhibitors. The increase of PG synthesis was accompanied with an undersulfation of constitutive glycosaminoglycan (GAG) chains (chondroitin sulfate and heparan sulfate chains) since the [35S]/[3H] ratio was reduced by about 20-25% in presence of IGF-1. Although the mechanism of hyaluronic acid synthesis was completely different from those of other GAG, IGF-1 also dramatically enhanced its production by PT cells.

Validation of a new system for androgen binding protein measurement.

A new technique that permits measurement of Androgen-Binding Protein (ABP) is validated by reproducibility, linearity and correlation studies. Using this apparatus allowing Scatchard plot analysis, it is also possible to measure association and dissociation rate constants. In addition, it is a very useful tool for a rapid screening of ABP binding capacity during a chromatographic stepwise purification.

[Effects of sodium molybdate on molecular forms of steroid receptors in human breast tumors. The value of high performance liquid chromatography (HPLC)]. / Effets du molybdate de sodium sur les formes moléculaires de récepteurs de stéroïdes des tumeurs mammaires humaines. Intérêt de la chromatographie liquide à haute performance (HPLC).

Estrogen, progesterone and glucocorticoid receptors from 124 breast cancer cytosols were studied by high performance liquid chromatography (HPLC), in comparison with sephadex chromatography and density gradient ultracentrifugation. Sodium molybdate was used as a receptor-hormone complex stabilizer, and as a tool allowing to study the different molecular forms of the receptors. In the presence of molybdate during the entire saturation process, more receptor sites were detected, without modification of equilibrium reaction kinetics. HPLC receptors analysis was rapid, with an high relative separation efficiency. Multiple molecular forms of steroid receptors were observed: five forms each for progesterone and glucocorticoid receptors, and six forms for estrogen receptors. The presence or absence of one of these forms emphasized the biological variability of the tumors and provides further questions or hormone-dependence.

A minicolumn apparatus for androgen-binding protein measurement.

An easy, rapid, and sensitive assay that permits measurements of androgen-binding protein (ABP) in tissue as well as in spent media from Sertoli cells is described; this method involves the specific binding of labeled dihydrotestosterone (DHT) to ABP. The apparatus holds 36 minicolumns loaded with a DEAE Bio-Gel matrix. A peristaltic pump is used for the free fraction elution, taking into account the extremely rapid rate of dissociation of the ABP-DHT complexes. This technique, which allows Scatchard plot analysis, has been used to measure the rates of association (5.15 X 10(5)M-1 S-1 and dissociation (21.32 X 10(-4) S-1; t 1/2 = 5.5 min): the ratio of these rate constants is in perfect agreement with equilibrium dissociation constants determined by Scatchard plot analysis (KD = 4-4.5 nM). The intraassay and interassay coefficients of variation are 5 and 8%, respectively. A good correlation (r = 0.98) is obtained with the standard method of steady-state polyacrylamide gel electrophoresis below a value of 250 micrograms cytosolic proteins/gel. This apparatus, which allows either the measurement of ABP in 12 samples (in triplicates) at a saturating concentration or the analysis of two Scatchard plots (each of 6 points), is also very useful for a rapid localization of ABP during chromatographic purification.

In vitro effects of growth factors on rat germ cell RNA synthesis and their modulation by Sertoli cell-secreted proteins.

In vitro rat germ cell RNA synthesis is influenced by growth factors. Basic fibroblast growth factor (0.1 to 100 ng/ml) increases [3H]uridine incorporation in round spermatids (RS) but not in pachytene spermatocytes (PS); this effect is potentiated by insulin (10 micrograms/ml) and blocked in the presence of Sertoli cell-secreted proteins (SCSP). Somatomedin C (0.1 to 100 ng/ml) exhibits a similar effect when used alone without an influence by SCSP. Transforming growth factor beta (0.1 to 10 ng/ml) acts on both cell types, but SCSP amplify this effect only in PS. These data suggest that growth factors synthesized in situ may play a role in the germ cell development and that their effects are modulated by SCSP.

Partial purification of androgen binding protein from bull epididymis.

An androgen binding protein (ABP), with an equilibrium dissociation constant of 4.2 nM and a molecular weight of about 100 kDa, has been purified from bull epididymal extracts using a four-step procedure. These preliminary results underline the main difficulties encountered in the purification of this protein present at a very low concentration (i.e. 50-fold less than in rat or rabbit epididymides). Ammonium sulfate precipitation is not a suitable step due to the formation, in presence of salt, of insoluble material leading to a loss of ABP. Lipids, particularly phospholipids, might be implicated in this phenomenon. Several steps, including anion exchange in batch followed by concentration, affinity chromatography and HPLC gel filtration allowed us to obtain a 7667-fold purified protein with a 9% yield.

Effects of transforming growth factor-beta 1, interleukin-1 alpha and interleukin-6 on rat Sertoli cell proteoglycan synthesis.

In the testis, growth factors and cytokines are synthesized by Sertoli cells and peritubular cells. In different cell types, these mediators are known to regulate the metabolism of extracellular matrix molecules, such as proteoglycans. In this study, we have tested the action of three of these mediators (IL-1 alpha, IL-6 and TGF-beta 1) on the proteoglycan synthesis of rat Sertoli cells. The proteoglycan synthesis was unchanged by IL-1 alpha, nor by IL-6 up to 20 ng/ml, during any maturation stage (14, 21 and 35 days). By contrast, Transforming Growth Factor-beta 1 (TGF-beta 1) enhanced Sertoli cell proteoglycan production from 21 day-old rats, with an optimal response at 1 ng/ml. Kinetic studies showed that the effect of TGF-beta 1 (1ng/ml) was higher after 24 hours of incubation. In presence or in absence of TGF-beta 1, a proteoglycan accumulation was observed in the extracellular compartment between 12 and 48 hours, but this factor did not modify the proteoglycan distribution between cell layer and medium. Furthermore, TGF-beta 1 increased proteoglycan anabolism at all Sertoli cell maturation stages studied but this stimulation was greater for the early maturation stages (14 and 21 days). On the other hand, proteoglycan catabolism was not modified by TGF-beta 1. In conclusion, TGF-beta 1 secreted by rat Sertoli and peritubular cells could modulate, in an autocrine/paracrine way the synthesis of one of the main extracellular matrix components.

Synthesis and distribution of rat Sertoli cell proteoglycans are modulated by linoleate and vitamin E.

In rat Sertoli cells, linoleate addition modified cell membrane fatty acid composition and changes depended on linoleate concentrations. In presence of the lowest 18:2 n-6 concentrations (2.5 and 7.5 microM), decrease in proteoglycan synthesis paralleled increase in n-6 linoleate-derived metabolites. At high concentration (21 microM), linoleate accumulated in membranes and level of n-6 linoleate-derived metabolites returned to basal value, without change in proteoglycan synthesis. Linoleate modified proteoglycan distribution in Sertoli cells by an increase in peripheral proteoglycans and a concomitant decrease in medium proteoglycans. Vitamin E (100 microM) did not alter fatty acid composition in control and linoleate-treated cells, but enhanced proteoglycan production. Furthermore, this agent counteracted linoleate-induced modifications in proteoglycan cell distribution.

Membrane associated proteoglycans in rat testicular peritubular cells.

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG). Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav = 0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column, they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.