Roland d'Argy

Transport and Subcellular Distribution of Nickel in the Olfactory System of Pikes and Rats

Occupational exposure to nickel by inhalation may result in impaired olfactory sense. Recent studies have shown that nickel is transported from the olfactory epithelium along the axons of the primary olfactory neurons to the brain. In the present study 63Ni2+ was applied in the olfactory chambers of pikes (Esox lucius) and the rate at which the metal was transported in the primary olfactory neurons was determined by beta-spectrometry. The results showed a wave of 63Ni2+ in the olfactory nerves, which slowly moved toward the olfactory bulbs. The maximal 63Ni2+ transport rate corresponding to the movement of the base of the wave front was found to be about 0.13 mm/h at the experimental temperature (10 degrees C). This rate of 63Ni2+ transport falls into the class of slow axonal transport. Radioluminography of tape sections of a pike given 63Ni2+ in the right olfactory chamber showed a selective labeling of the right olfactory nerve. The subcellular distribution of 63Ni2+ in the olfactory nerves and the olfactory epithelium of the pikes was studied in tissues subjected to homogenizations and centrifugations, and these methods were also used to examine the subcellular distribution of 63Ni2+ in tissues of the olfactory system of rats given the metal intranasally. It was found that the 63Ni2+, in both the pike and the rat, was present in the cytosol and also in association with various particulate cell constituents. Gel filtrations of the cytosols showed that the 63Ni2+ mainly was eluted at a Ve/Vo ratio corresponding to a MW of about 250. The same coefficient was obtained in gel filtrations performed with 63Ni2+ mixed with histidine in vitro. It is likely that the cytosolic nickel may be bound to histidine or possibly to other amino acids which are similar in size to histidine. Additionally, in the olfactory tissues of the rat the 63Ni2+ was partly present in the cytosol in association with a component with a MW of about 25,000. It is concluded that (i) 63Ni2+ is transported in the primary olfactory neurons by means of slow axonal transport, (ii) in this process the metal is bound to both particulate and soluble cytosolic constituents, and (iii) the metal shows this subcellular distribution also in other parts of the olfactory system.

Teratological studies on the TCDD congener 3,3',4,4'-tetrachloroazoxybenzene in sensitive and nonsensitive mouse strains: evidence for direct effect on embryonic tissues.

The teratogenicity of 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB), a TCDD congener, was studied in Ah-responsive (C57BL and NMRI) and non-responsive (DBA/2J and AKR/NBom) strains of mice. In the responsive strains, the TCAOB produced cleft palate and hydronephrosis in 50–90% of the offspring at a dose level of 6–8 mg/kg b.w. in the absence of apparent maternal toxicity. Day 11 was shown to be the day of highest sensitivity (palatal closure occurs at day 14) in the C57BL strain. Higher doses (16 mg/kg b.w.) produced high rate of fetal death both in responsive (C57BL; 60%) and non-responsive (DBA; 40%) strains. These doses induced cleft palate in 95% of the surviving C57BL fetuses but failed to do so in the DBA strain. The non-sensitivity of the DBA and AKR strains appeared to segregate as a dominant trait. Backcrosses between NMRI x DBA F1 generation and NMRI showed an intermediate sensitivity. It was shown that the genotype of the embryo was of ultimate importance for the development of cleft palate. There appeared however to be an additional host (maternal) factor as well, because the offspring of NMRI females mated with NMRI x DBA F1 males showed a higher rate of cleft palate as compared to those of the crossing between NMRI x DBA F1 females and NMRI males. Light and scanning electron microscopy indicated that the apical epithelial cells of the secondary palates failed to follow the normal pattern of programmed cell death, suggesting a similar mechanism of pathogenesis as previously described for TCDD.

COMPUTER-ASSISTED QUANTIFICATION AND IMAGE PROCESSING OF WHOLE-BODY AUTORADIOGRAMS

A computerized image-processing system especially adapted for analysis of whole-body autoradiograms has been developed. It consists of commercially available standard components, including a black-and-white video camera, a microcomputer, and graphics equipment. The lower performance of the hardware has been compensated for by more flexible software. When the system was calibrated, special attention was paid to local variations in the measuring system in different parts of the picture. Utility programs for the manipulation of contrast, pseudocoloring, and image enhancement, etc., are available. Some programs have been especially designed to comply with specific problems and demands related to different autoradiographic applications. A program displaying the density histogram for an area of interest is particularly useful for the quantitation of whole-body autoradiograms. It allows the operator to select interactively a range of densities. Image elements (pixels) corresponding to the densities in this range are shown in red on the monitor, and their average true density is calculated. This procedure permits the marking and analysis of delicate structures on autoradiograms. Other programs allow a picture, stored in memory, to be rotated or translated, and two pictures to be superimposed for comparison. Various applications of using image analyses in whole-body autoradiography are presented and illustrated.

Teratogenicity of 2,3,7,8-tetrachlorodibenzofuran in BXD recombinant inbred strains

A series of recombinant inbred strains called BXD [produced from a cross between C57BL/6J (B6) and DBA/2J (D2)] were given single i.p. doses of 0.6 mg/kg 2,3,7, 8-tetrachlorodibenzofuran (TCDBF) on day 12 of gestation. The uteri were examined in late gestation with respect to resorptions and fetal death, and fetal malformations. The strains of the B6-type with respect to Ah-locus (Nos. 5, 6, 8, 11, 12, 14, 16 and 29) that are Ah-responsive, exhibited cleft palates in 80-100% of all fetuses, while hydronephrosis occurred at a rate of 20-70%. These two types of malformation are well recognized from earlier experiments with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its structural analogues, including TCDBF. In the strains of D2-type with respect to Ah-locus (Nos. 2, 15, 19, 21, 22, 24, and 31), which are Ah-nonresponsive, no cleft palates occurred. One strain (No. 2) had a few (17%) fetuses with hydronephrosis. The frequency of fetal deaths and resorptions were relatively low, but slightly higher among B6-strains than D2-strains. The results indicate an association between the genes producing malformations by TCDBF and the Ah-locus.

Whole-body autoradiography using 11C with double-tracer applications

This paper reports that it is possible to obtain good autoradiographic pictures of a 11C-labelled compound ([11C]methionine) in spite of the short half-life of 20 min by modifying the whole-body autoradiographic (WBA) technique. Double radionuclide autoradiography was performed by utilizing the great difference between the half-lives of 11C and 14C or 35S. The first exposure resulted entirely from 11C, and the second exposure the day after from 14C or 35S, respectively. Satisfactory blackening of the film was obtained by a mean radioactivity of 1.6 X 10(6) disintegrations/cm2 during the exposure. The 11C-WBA technique apparently provides a useful tool in distribution studies on animals, which is valuable in the interpretation of positron emission tomography images of the same 11C-compound.

The in vivo uptake of tritiated thymidine as a potential short-term test of toxic effects of polycyclic aromatic hydrocarbons in different organs

A rapid in vivo test for toxicity is described where the test substance is allowed to distribute and metabolize in the intact mouse. Quantitative data are provided on the effect of 3 different polycyclic aromatic hydrocarbons on the DNA turnover in various organs, measured as the incorporation of tritiated thymidine. In accordance with previously reported carcinogenic potencies, 7,12-dimethylbenz[alpha]anthracene (DMBA) was more potent in inhibiting the thymidine incorporation than benzo[alpha]pyrene (B[alpha]P). The inhibitory effect was most pronounced in spleen, lung, pancreas, small intestine and kidney, resulting in a decrease of incorporated activity with up to 80%. There was no evidence for the existence of specific target organs for DMBAs effects on thymidine incorporation as indicated by an inhibitory action in all organs studied. A decreased thymidine incorporation after administration of DMBA could also be demonstrated with whole-body autoradiography. The inhibitory effect of B[alpha]P was most pronounced in thymus, spleen, small intestine and testis, the average decrease of incorporated activity being more than 40% after 48 h. Contrary to the wide action of the above mentioned polycyclic aromatic hydrocarbons, an equimolar dose of anthracene lacked significant effects on the various organs.

Comparative double-tracerwhole-body autoradiography: Uptake of 11C−, 18F− and 3H-labeled compounds in rat tumors

The uptake of various labeled compounds by tumors was studied by double-tracer whole-body autoradiography (DTWBA) in rats. Each animal carried two types of tumors: mammary carcinomas and the Walker 256 carcinosarcomas. The markers used were [18F]- and [3H]fluorodeoxyglucose (glucose utilization), [3H]thymidine (cell proliferation), [11C]methionine (amino acid metabolism) and [11C]- and [3H]toremifene (estrogen-receptor-avid agents). In each experiment, the distribution of a substance labeled with short-lived radionuclide (11C or 18F) was compared with that of another substance labeled with a long-lived nuclide (3H). Quantification was done by combining computerized image analysis of the autoradiograms with liquid scintillation counting of punched tissue pieces obtained from the cryosections. The relationships between the uptakes of the various radiopharmaceuticals were recorded in tumors and normal tissues. The dynamics of [18F]fluorodeoxyglucose and [11C]methionine were determined in tumors and some selected tissues by positron emission tomography (PET). The uptake rate between fluorodeoxyglucose and thymidine in the mammary tumor was five times higher than the ratio in the Walker tumor. The corresponding figure for FDG/methionine was four times. Thymidine, compared with methionine, was twice as efficient. Thus, the mammary tumors were best imaged with FDG or thymidine. The non-steroid antiestrogen toremifene was taken up in very low amounts by these tumors. By DTWBA, experimental tumors may serve as their own control.

Distribution of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in experimental animals studied by postiron emission tomography and whole body autoradiography

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a selective potent neurotoxin which has induced a syndrome similar to parkinsonism both in man and in monkeys. At autopsy degeneration of pigmented nerve cells in the pars compacta of the substantia nigra has been confirmed. The regional distribution of intravenously administered 1-(11C-methyl)-4-phenyl-1,2,3,6-tetrahydropyridine (11C-MPTP) in the brain of Rhesus monkeys was studied by positron emission tomography and the whole body distribution in mice was documented by autoradiography and by impulse counting of selected tissues. A very rapid and high uptake of 11C-MPTP derived radioactivity was seen in areas corresponding to striatum and midbrain, including the substantia nigra area. No elimination from these regions was seen during the study period of 2 h. The uptake was in the order of 7-8 times the homogenous distribution of the radioactivity in the monkey. The uptake was generally high also in other regions of the brain, but there some elimination could be distinguished. Pretreatment of the monkey with spiperone, a selective dopamine receptor antagonist, did not alter uptake nor the kinetics of the 11C-MPTP derived radioactivity. Thus 11C-MPTP does not have a high affinity for postsynaptic dopamine receptors. A remarkably high uptake of 11C-MPTP derived radioactivity was seen in the eye of the monkey. The selective uptake of radioactivity in the eye was also confirmed in pigmented but not in albino mice. The melanin affinity of MPTP may cause high intracellular concentrations of the compound or its metabolites in the melanin containing nerve cells in substantia nigra, which may explain the serious vulnerability of these neurons to MPTP.

Tissue Disposition of Carbon Disulfide. II. Whole-Body Autoradiography of 35S- and 14C-labelled Carbon Disulfide in Pregnant Mice

Occupational exposure to carbon disulfide (CS2) has been associated with an increased rate of spontaneous abortions. Animal experiments have shown that CS2 is embryotoxic and teratogenic. In the present study, the embryonal and foetal distribution of CS2 and its metabolites was studied after administration of 35S- or 14C-labelled CS2 to pregnant mice in different stages of gestation. CS2 and its metabolites passed the placenta at all stages of gestation. High levels of metabolites of CS2 were registered in the embryonic neuroepithelium. In mid and late gestation CS2 itself accumulated in the cerebrospinal fluid (CSF) of the brain. 14C-labelled metabolites of CS2 showed affinity for bone and were retained in the liver even at long survival times (24 hours). These localizations may be of significance for some of the reported teratogenic effects of CS2, such as hydrocephalus, ossification defects and foetal liver injury, and support the idea that CS2 and/or its metabolites are embryotoxic and teratogenic by acting directly on embryonal and foetal structures.