Nuclear import of cellular retinoic acid-binding protein type I in mouse embryonic cells.

Using confocal microscopy we show that cellular retinoic acid-binding protein type I (CRABP I), expressed in several embryonic cell types, displays a compartmentalized subcellular distribution. The protein was excluded from the nucleus in some cells, while in others it accumulated in the nucleus. In the rat cerebellar cell line ST15A, which expresses CRABP I, the protein was found in the cytoplasm with a prominent nuclear exclusion. Addition of retinoic acid to embryos in vivo and to ST15 A cells in vitro did not affect the localization of the protein. Localization of CRABP I and CRABP I fused to a nuclear localization signal expressed in transfected cells, suggested that cell-specific factors may regulate nuclear import of CRABP I. The potential role of a CRABP I-controlled nuclear import of retinoic acid is discussed.

Oil-induced arthritis in DA rats: tissue distribution of arthritogenic 14C-labelled hexadecane.

As part of the study of the pathogenesis of oil-induced arthritis in DA rats, the tissue dissemination of arthritogenic oil labelled with 14C has been determined. Rats received labelled hexadecane in arthritogenic doses by the intradermal route and were killed at 1 and 6 h, 2, 10, 14, 18 and 27 days after injection. Whole-body autoradiography was performed and localization of radioactivity in different organs was investigated. With the exception of the injection site, the lymph nodes showed the highest content of radioactivity throughout the study. Radioactivity could be seen in the popliteal, inguinal and axillary lymph nodes. Detailed examination of lymph nodes revealed at 1 h radioactivity in the subcapsular sinus of the lymph node. By 10 days the activity had spread to the cortex and paracortex, apart from the subcapsular sinus. The activity in other lymphoid organs, such as the bone marrow and spleen, was more transient, with a peak at 2 days. Oil disseminating to joints was not prominent: very little radioactivity was observed in the knee joints of both non-arthritic and arthritic animals. From these data it is suggested that the adjuvant oil exerts its major proarthritogenic activity in the lymph nodes rather than directly in the joints.

In vivo and in vitro toxicity of fractionated fish lipids, with particular regard to their content of chlorinated organic compounds.

Six different lipid matrices (the intact lipid (IL), four lipid fractions with different polarity, and the free fatty acids (FFAs) obtained by hydrolysis of the triacylglycerol (TAG) containing fraction) were obtained from salmon (Salmo salar) and eel (Anguilla anguilla), each collected at a contaminated and a comparatively uncontaminated catch site along the coast of Scandinavia. The lipid matrices were studied in toxicological test systems representing various biological functions of different organ systems from several species and trophic levels. The results were evaluated with particular respect to the concentrations of extractable organically bound chlorine (EOC1) in the matrices tested. In some test systems, the specimens with a higher EOC1 concentration appeared to be more toxic. For example, the TAG containing fraction (F2) from Idefjord eel, having a higher EOC1 content than F2 from Oslofjord eel, reduced the number and hatchability of eggs laid by zebrafish. Both IL and F2 of Idefjord eel increased mortality and reduced the oxygen/nitrogen-ratio in blue mussels. Non-polar compounds (F1) from Bothnian Sea salmon induced 7-ethoxyresurofin O-deethylase (EROD) activity in rainbow trout hepatocytes, whereas F1 from Senja salmon did not. F1 from Bothnian Sea salmon also reduced the number of T-cells in foetal mouse thymus analagen in vitro compared with the cell number in anlagen exposed to F1 from Senja salmon. A positive correlation between EOC1 concentration and test response was found for EROD activity in rainbow trout hepatocytes and for ATP-leakage in Erlich ascites tumour cells when testing the phospolipid containing fraction (F4). However, in most test systems the fish oils, irrespective of EOC1 content, were of low toxicity, and the observed effects need to be verified in future studies.

Retinoid-binding proteins in craniofacial development.

Cephalic neural crest cells are known to form the frontonasal mesenchyme and contribute to the mesenchyme of the visceral arches. Retinoids affect neural crest cells and their derivatives during development, and thus cause craniofacial, thymus, and conotruncal heart malformations. In addition, retinoids induce malformations of the central nervous system (CNS). Retinoic acid (RA) and its congeners accumulate in a saturable manner in neural crest and neural crest-derived cells, in the hindbrain, and the spinal cord of mouse embryos. Cellular retinoic acid-binding protein (CRABP) was localized by immunohistochemistry in the same areas as were the labelled RA congeners. Thus, CRABP and RA congeners were found in the transitional zone between surface ectoderm and neuropeithelium, from where neural crest cells are known to emanate (day 8 1/2). Later, specific labelling was found in the frontonasal mesenchyme and in the visceral arches. Also in the trunk, neural crest cells were labelled. In CNS, strong staining was seen in the rhombomeres (especially numbers 4-6) of the hindbrain and in the spinal cord. Retinol and cellular retinol-binding protein (CRBP) were more evenly distributed, with exception of surface ectoderm, epithelium of gut, and myocardium, where CRBP was specifically expressed. These findings are discussed in relation to the differential expression of nuclear RA receptors and homeobox genes in the craniofacial region and in the hindbrain. It is possible that RA is important for the normal pattern formation in these regions and acts as a morphogen as previously proposed in limb development.

Marked accumulation of valproic acid in embryonic neuroepithelium of the mouse during early organogenesis.

Valproic acid, an antiepileptic drug, causes neural tube defects in mice and man. 14C-labeled valproic acid (sodium-salt) was administered to pregnant mice on days 8 and 9 of gestation (period of high sensitivity in regard to formation of neural tube defects in this species). Two dose levels of valproic acid (1 and 400 mg/kg) were used; in each case the total radioactivity administered was the same: 400 microCi/kg or 14.7 MBq/kg. Autoradiography combined with computerized densitometry revealed that in low-dose animals most of the radioactivity was confined to maternal liver and kidney, while at high doses more activity was observed in soft tissues and fluids, including amniotic fluid. In the embryo, the neuroepithelium showed the highest concentration, irrespective of dose and survival interval (30 min, 3 h, and 6 h). Upon administration of the high dose, up to five times more radioactivity (approximately 2,000 times more valproic acid) was recovered in embryonic tissues than after the low dose. It is concluded that high doses of VPA saturate the capacities of metabolism, excretion, and protein binding in the maternal organism, resulting in a higher proportion of the dose reaching the embryo, allowing more of the drug to be accumulated by the target organ, the neuroepithelium.

Selective incorporation of thiouracil into murine metastatic melanomas.

The uptake and retention of 14C-thiouracil and 125I-thiouracil in small lung metastases of B16 murine melanoma was studied in beige mice injected intravenously with melanoma cells. By impulse counting of excised tumor and organ pieces, a high concentration of radioactivity was found in the lung metastases, as compared to normal tissues. The highest tumor/organ concentration ratios appeared 24 h after injection of the radiolabeled thiouracil. A separate autoradiographic study on the disposition of 14C-thiouracil in mice with melanoma metastases confirmed the impulse counting results and also showed the absence of any other site of retention of radioactivity except for hair follicles and to some extent the thyroid. The selective uptake of 14C- and 125I-thiouracil in melanomas depends on their acceptance as false melanin precursors, making them specific markers for growing melanin. The results indicate that radiolabeled thiouracil may be useful for clinical diagnosis and, possibly, therapy of malignant melanotic melanomas.

Preferential localization of 3H-pentosanpolysulphate to the urinary tract in rats.

Endogenous glycosaminoglycans probably have a protective effect in the urinary tract, e.g. against stone formation. The synthetic sulphated polysaccharide pentosanpolysulphate (PPS) has been suggested to exert a similar protective effect e.g. by inhibition of crystallization and bacterial anti-adhesion. We have studied the distribution in rats of tritium-labelled PPS. Chromatography showed this material to contain two distinct peaks with approximate molecular weight around 2.700 (60-70%) and 1.000 (30-40%) daltons. PPS was administered orally and intravenously (5 mg/kg b.wt.) to Sprague-Dawley rats, which were killed 1 and 4 hours later, respectively, and subjected to whole-body autoradiography. Autoradiograms of sections from intravenously injected rats showed an extensive distribution of radioactivity in the whole animal, with a notable labelling of connective tissues, while bone and cartilage had low activity. There was upper intestine activity, suggesting some hepatic excretion. The most conspicuous finding, however, was the high concentration in urine and a preferential localization of activity corresponding to the lining of the urinary tract (pelvis, ureter, and bladder). The distribution was similar, but the activity lower after oral administration. In one experiment, PPS was applied intravesically under anaesthesia, with and without epithelial destruction caused by instillation of 0.4 M HCl. After vigorous rinsing, with saline, the radioactivity was still retained in the bladder wall. In other intravenous experiments, the bladder was extirpated, everted and rinsed in saline or urea of increased osmolality. High amount of radioactivity could be rinsed off by 0.5 M saline. Chromatography of the rinsing solution showed presence of both fractions of PPS previously found in the injection solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturable accumulation of retinoic acid in neural and neural crest derived cells in early embryonic development.

Retinoic acid (RA) binds to a cytosolic protein distinguishable from the cellular retinol (R) binding protein. Recent studies, showing an influence by R and RA on genomic expression, suggest an interaction with the cell nucleus mediated by the specific binding proteins in a manner resembling that of steroid hormones. RA can irreversibly stimulate in vitro differentiation of teratocarcinoma cells and support early embryonic development in vitamin A depleted animals. This study demonstrates a saturable, highly specific and regional accumulation of RA in the neuroepithelium and developing CNS that occurs in early but not in late fetal development in the mouse. The results suggest that a binding protein, or some other cellular mechanism for accumulation of RA is expressed in the neural cells only during restricted periods of development. High levels are recorded also in regions where cranial neural crest cells are known to migrate, and later in the visceral arches and maxillary areas, the mesenchyme of which is known to be partly derived from migrating cranial neural crest cells. The specific accumulation of RA in embryonic neural and cranial neural crest cells is in line with animal experiments and human clinical data, showing that retinoids specifically impair CNS, eye, ear, and facial development.

Renal handling and effects of [3H]digoxin and interactions with quinidine in the avian kidney.

Renal handling and effects of tritium digoxin and interactions with quinidine in the avian kidney were studied using a modified Sperber technique. Results showed that tritium digoxin was extracted at the peritubular side of the nephron in a process competitively inhibited by increasing amounts of unlabelled digoxin. Light microscope autoradiography showed distinct concentrations of silver grains only over distal tubules in the injected kidney. Inhibition of the proximal tubular transport systems for organic anions and cations, respectively, did not change extraction. Addition of quinidine to the injection solution up to an estimated concentration of 1.4 X 10(-5) M in systemic blood significantly lowered 1 min peritubular extraction of tritium digoxin. However, when the amount of quinidine was further increased, extraction of tritium digoxin augmented significantly. Tritium recovery in urine after renal portal bolus injection of tritiated and unlabelled digoxin already showed a distinct ipsilateral peak 2 min after injection with an equally distinct peak of ipsilateral sodium excretion appearing 1 min later. When quinidine was added to the bolus ipsilateral tritium recovery in urine (0-7 min) was halved, with the true tubular excretion fraction (TTEF) lowered by two-thirds, but without changes in the magnitude of ipsilateral natriuresis. Contralateral natriuresis increased more than four-fold with quinidine in the bolus in spite of unchanged tritium recovery in the urine. Thus, our results show tritium digoxin to be extracted from peritubular blood through a specific process probably localized to the distal nephron of the avian kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance of different lymphoid cells in synovial tissue and in peripheral blood during the course of collagen II-induced arthritis in rats.

The involvement of different sets of lymphoid cells in the development of collagen II-induced arthritis in rats was studied by means of immunohistochemical analyses on frozen sections of tissue from joint biopsy specimens taken at different phases of arthritis development. Particular attention was paid to cells involved in early pannus formation. Accumulation of anti-Ia-reactive cells close to the cartilage surface was seen early in the development of pannus, and the anti-Ia reactive cells could in later phases be seen infiltrating cartilage and crowding bone surfaces at sites of marginal erosion. With the help of monoclonal anti-T-cell subset antibodies and rabbit anti-rat immunoglobulin antiserum, it was demonstrated that synovial infiltration of T lymphocytes, particularly W3/25-reactive T "helper" cells, occurs very early in the development of arthritis, whereas a moderate increase of Ox 8-positive 'suppressor/cytotoxic' T cells and a small number of B cells and plasma cells are seen later in the course of the disease. Levels of Ia-expressing cells and of T cells belonging to different subsets were recorded in peripheral blood by means of immunocytochemical analyses on cell smears; no significant deviations from normal levels were seen during the development of arthritis.

The role of receptors in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity.

There is good evidence that the Ah-(TCDD-) receptor plays a role in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its congeners. TCDD and other chlorinated aromatic hydrocarbons with chlorine atoms in lateral positions (2,3,7,8-tetrachlorodibenzofuran, 3,3',4,4'-tetrachloroazoxybenzene, 3,3',4,4'(5,5')-tetra(hexa)chlorobiphenyl), all bind to the receptor and show a similar pattern of toxicity, although there is a wide range in potency. The Ah-receptor is viewed as the major product of the regulatory gene of the Ah-locus in the mouse. Several of the toxicities of TCDD and congeners (teratogenesis, thymic involution and hepatic porphyria) have been shown to segregate with the Ah-locus. In vitro studies using keratinizing cells or fetal thymus organ culture have shown a good correlation between activity as ligands of the receptor and toxicity for the compounds discussed. The great differences in toxic potency of these compounds in vivo may therefore be a result of variation in rate of metabolism and excretion rather than differences in affinity for the Ah-receptor. The physiological role of the Ah-receptor is discussed, whether it has developed as a response to exposure to toxic substances in the environment, as a means of induction of P-450-dependent polysubstrate mono-oxygenase activities in order to make those substances more liable for excretion--or is there a physiological ligand? TCDD has a long half-life in the body, and a sustained competition for binding to the receptor between TCDD and a ligand of importance for normal cell functions may result in toxicities such as the wasting syndrome. This tentative ligand could be of varying importance in different species, which might explain the great variation in sensitivity between species, the hamster being about 5000 times less sensitive than the guinea pig.

Fetotoxicity of inorganic mercury in the mouse: distribution and effects on nutrient uptake by placenta and fetus.

Different aspects on the fetotoxicity of HgCl2 have been studied in vivo and in vitro and have been compared to earlier results with CdCl2, which is known to cause placental necrosis and fetal death. Hg has to be given in higher doses (20-25 mg/kg body weight) than Cd (3-4 mg/kg body weight) to cause fetolethality in late pregnancy in the mouse. It does not cause the typical signs of placental damage (congestion and bleedings) as does Cd. At a dose level of 15 mg/kg body weight, Hg reaches more than 10 times higher concentrations (approx. 5 times higher on a molar basis) in the fetus, while Cd (4 mg/kg body weight) on the other hand appears in the placenta at a double molar concentration as compared to Hg. In a chick, limb bud mesenchyme cell culture differentiating into chondrocytes, Hg and Cd were about equally effective in inhibiting cartilage-specific staining by alcian blue (ED50 approximately 1 microM). Due to the strong accumulation of Hg and Cd in the placenta, the effects of Hg on the placental and fetal uptake of four nutrients was studied. Hg caused a dose-dependent decrease in fetal radioactivity as compared to controls 4 hours after administration of 57Co-vitamin B12 to the mother. However, this effect was not as marked as for Cd. Hg, on the other hand, decreased fetal radioactivity after 14C-alpha-aminobutyric acid more than Cd did. Both elements decreased the fetal uptake of 65Zn, probably due mainly to a concomitant decrease in maternal serum concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cadmium on the placental uptake and transport to the fetus of nutrients.

Cadmium (Cd) is known to produce malformations, growth inhibition of the fetus, and placental necrosis in rodents at higher doses (3-4 mg/kg body wt). We studied the influence of Cd in various doses (0.5-4 mg/kg body wt) and at different survival intervals on the placental uptake and transfer to the fetus of vitamin B12 (vit. B12), zinc (Zn), alpha-aminobutyric acid (AIB), and deoxyglucose (DOX). These were chosen to represent various mechanisms of membrane transport. We show that vit. B12, which is accumulated in the placenta by a presumed receptor-mediated mechanism, is most easily disturbed by Cd. Thus, a significant decrease in transfer to the fetus was seen already 1 h after a high dose (4 mg/kg body wt) of Cd, and also at longer intervals (24 h) after low doses (0.5 mg/kg body wt). The transport of Zn (chemically similar to Cd) was also disturbed, but its inhibition was probably due in part to a decreased maternal serum concentration. The transport of AIB and DOX was largely unaffected. We conclude that inhibition of nutrient transfer to the fetus may be the underlying mechanism of growth retardation and possibly of the malformations produced by Cd. Vitamin B12 may be a sensitive indicator of early and subtle disturbances of placental function, not only for Cd but also for other chemicals suspected of causing placental disturbances.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced cleft palate in the mouse: evidence for alterations in palatal shelf fusion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes a high percentage of cleft palate in fetuses when administered during organogenesis in certain strains of mice including the C57BL/6J, but not in certain other strains (AKR/J). The purpose of the present study was to examine various biochemical and morphological aspects of TCDD-induced changes in the developing palatal shelves. Our results indicate that when TCDD (100 micrograms/kg) was given on individual days between days 8 and 10 of gestation, a high percentage of cleft palate was observed. Receptors specific for TCDD were detected in the C57BL/6J but not AKR/J palatal shelves. The amount of TCDD receptors is highest in the palatal shelves on day 13 as compared to other embryonic tissues including the liver. Examination of cryostat sections taken from embryos during the time of palatal elevation and fusion demonstrated that TCDD does not interfere with growth, elevation, or initial contact of the palatal shelves, but does interfere with firm adhesion and/or degeneration of the medial epithelial cells. Our results suggest that TCDD exerts a direct effect on the embryonic palatal shelves which results in formation of cleft palate.

Thioamides as false melanin precursors: studies in murine melanomas.

Melanotic melanomas show a high rate of melanin synthesis. Foreign substances that are accepted as precursors in the formation of melanin may therefore be useful in the diagnosis and therapy of malignant melanotic melanomas, if labelled with suitable radionuclides. We have earlier reported that 2-thiouracil is incorporated in melanotic melanomas, apparently as a false melanin precursor. In the present study it is shown that methimazole and 5-iodo-2-thiouracil are as well accepted as melanin precursors. 5-Iodo-2-thiouracil is of special interest, since iodine has many clinically useful radioisotopes. The chemical properties that characterize substances which are incorporated as false precursors into melanin are discussed. A free sulfur ligand of the thioamides (2-thiouracil, 5-iodo-2-thiouracil, methimazole and thiourea are all incorporated into melanin) seems to be essential and the link between these substances and the melanin. Uracil (which lacks sulfur) and 2-benzylthiouracil (where the sulfur is blocked with a benzyl group) do not attach to melanin. Our conclusion therefore is that the thioureylene structure is the smallest common molecular fragment of the false melanin precursors.

Accumulation of 125I-labelled thiouracil and propylthiouracil in murine melanotic melanomas.

We have shown that thioamides are incorporated as false precursors into melanin during its synthesis. To be clinically useful in the diagnosis or therapy of melanotic melanomas, they would have to be tagged with an appropriate isotope or possibly a cytotoxic moiety. 125I-Thiouracil (125I-TU) is here shown to be accumulated in the melanin of melanotic melanomas transplanted into mice in a similar way as is 14C-thiouracil (14C-TU). 125I-TU gives tumour/liver and tumour/muscle ratios up to 22 and 778 respectively, at 4 days after administration. 125I-TU is accumulated by melanoma cells in vitro more effectively than 14C-TU (125I-TU/14C-TU, 2.7), while the in vivo accumulation into melanomas is slightly lower for 125I-TU as compared to 14C-TU (125I-TU/14C-TU, 0.35). This appears to be due to a partial deiodination (less than 14% of the dose within 4 days) and probably a more rapid excretion of 125I-TU or its metabolite(s). The accumulation of radioactivity in the thyroid can essentially be eliminated by pretreatment with potassium iodide and/or thyroxine. 125I-Propylthiouracil is also accumulated in melanotic melanoma cells in vivo and in vitro, but at a lower level than in 125I-TU and 14C-TU.

Disposition of chemicals in the developing embryo and fetus.

The uptake of foreign compounds and their distribution in the embryo and fetus at different stages of gestation are reviewed. In particular studies performed by means of autoradiography are discussed. It is shown that a number of compounds may be almost completely blocked by the placental structures early in gestation, although they are foreign compounds. In late gestation often one or a few organs may accumulate a particular compound; this may be due to metabolism, affinity for tissue components, etc. In some cases the site of accumulation of tissue binding of a drug has correlated well with the teratogenic or carcinogenic effects.

A new melanoma seeker for possible clinical use: selective accumulation of radiolabelled thiouracil.

In a previous report we have shown that a few substances, especially thiouracil, are incorporated as false precursors into melanin during its synthesis. In the present investigation, we have intensified our studies on the incorporation of thiouracil into melanotic melanomas. Firstly, the distribution and retention of both 14C- and 35S-labelled thiouracil in mice with transplanted melanomas were studied. A high and selective accumulation was found in the melanotic tumours. The concentration in the rest of the body was low, with the exception of the thyroid gland. Secondly, melanoma-bearing mice were given increasing doses of thiouracil, and cultured melanoma cells were exposed to different concentrations of thiouracil, to investigate the relation between dose and uptake in melanomas and melanoma cells, respectively. A relatively linear increase in uptake with dose was found, indicating that the melanin incorporation of thiouracil is non-saturable up to subtoxic levels.

Incorporation of thiouracil and some related compounds into growing melanin.

Several drugs, mainly polycyclic amines, are accumulated in melanin-containing tissues. They are bound to preformed melanin (both in vivo and in vitro). Thiouracil is accumulated into melanin according to another principle: It is incorporated as a false precursor during melanin formation. It is thus taken up only in growing melanin, e.g. in the eye of pigmented mouse foetuses or in melanomas. The acceptance of a foreign substance during the formation of a foetal tissue seems theoretically important. We are also interested in the practical viewpoint of using false melanin precursors as selective melanoma seekers. Some related substances are therefore compared with respect to incorporation into growing melanin. The thiouracil uptake in the ocular melanin of a 5 day old mouse was 276 times higher than that of a 3 month old mouse based on weight units of melanin. Thiourea is incorporated in growing melanin as well but also binds slightly to preformed melanin. Uracil and fluorouracil showed no specific uptake into growing melanin. It thus seems as if the sulfur is essential for the incorporation into the melanin polymer. 35S-thiouracil and 2-thio(2-14C)urcal showed the same high uptake, indicating that at least part of the uracil moiety is incorporated together with the sulfur. There seems to be a relation between the property to be incorporated into melanin and the tyrostatic activity. Both in the formation of melanin and thyroid hormones, tyrosine is the physiological precursor and both reactions are catalyzed by oxidizing enzymes. Properly labelled thiouracil derivatives seem to be promising melanoma seekers for diagnostic and radiotherapeutic purposes.

Distribution of 35S in mice after oral administration of alpha-dithioacetamidinium chloride. An autoradiographic investigation.

The distribution of 35S in mice has been investigated by whole-body autoradiography after oral administration of the 35S labelled S-75 (2,2'-Dithiobis(N-[(1-adamantyl)-methyl]-acetamidine)dihydrochloride). The substance was rapidly absorbed, and the highest concentration occurred in the liver and kidneys. During the highest radiation protective activity (after about 45 min) a substantial concentration was found in the red pulp of the spleen. This supports previous findings that S-75 has its most marked protective effect on the splenic haemopoiesis. Previously, it was shown that cysteamine is more evenly distributed in the body than S-75, which is in agreement with its more generalized protective effect than S-75.