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Fate and distribution of radioactive sodium diethyldithiocarbamate (Imuthiol®) in the mouse
Int. J. lmmunopharmac., Vol. 8, No. 8, pp. 859-865, 1986. Printed in Great Britain.
0192 0561/86 $3.00+ .00 International Society for lmmunopharmacology.
FATE AND DISTRIBUTION OF RADIOACTIVE SODIUM DIETHYLDITHIOCARBAMATE (IMUTHIOL ®) IN THE MOUSE JEAN-MAURICE GUILLAUMIN,ALAIN LEPAPE and GI~RARDRENOUX* Laboratoire d'Immunologie, Facult6 de M6decine, 37032 Tours Cedex, France (Received 17 October 1985 and in finalform 26 February 1986)
- - The distribution of 35S in mouse tissues has been investigated by radioactivity counts and by autoradiography after the intravenous injection of 35S-labeledimuthiol (sodium diethyldithiocarbamate). Radioactive Imuthiol is selectivelylocalized on liver, thymus and brain neocortex, most likely as the methyl ester, within minutes after dosing. Lung or white brain matter did not fix the labeled thiol. Blood, kidney and guts show rapid elimination patterns, and can be considered as passage organs which did not fix lmuthiol. The findings are consistent with the immunopharmacological data which demonstrate that Imuthiol exerts its T-cell recruiting and activating influence through a multi-step pathway involving the brain neocortex, the thymus and the liver.
Abstract
As an immunotherapeutic agent, Imuthiol® (purified sodium diethyldithiocarbamate), specifically recruits and activates T cells, as well as NK activity, and does not directly affect B cells and macrophages (Renoux & Renoux, 1979, 1980, 1983; Renoux, 1982; Pompidou, Renoux, Guillaumin, Mac6, Michel, Coutance & Renoux, 1984). Imuthiol can replace the inductive signals emitted by the left brain neocortex and it controls the synthesis of a T-cell specific liver factor, hepatosin (Renoux, Bizi6re, Renoux & Guillaumin, 1983; Renoux, Renoux, Bizi6re, Guillaumin, Bardos & Degenne, 1984). It increases the in vitro liver synthesis of hepatosin (Renoux, Renoux & Guillaumin, 1982; Renoux, 1984). The biological attributes of Imuthiol have been recently reviewed (Renoux, 1982). These include: destruction of neoplastic cells, parasites, fungi and bacteria at doses inactive on normal mammalian cells; a detoxifying influence against chemical carcinogens or agents toxic for the liver; an antistress effect through the regulation of dopamine/3 hydroxylase; an anti-inflammatory activity suggested by its influence on superoxide dismutase levels was recently confirmed (Renoux, Giroud, Florentin, Guillaumin, Degenne & Renoux, 1986). All these activities were thought to be associated with the
chelating and lipophilic properties of sodium diethyldithiocarbamate. A study was undertaken to reevaluate the distribution of the agent in the organs of mice by means of 35S-labeled Imuthiol in an attempt for a better understanding of the apparently complex mode of action of the agent. The data reported herein give evidence for a preferential localization of 35S on liver, thymus and brain neocortex. The findings fit in with the conclusions of the biological studies, which suggest that these three organs are involved in the multi-step pathway leading to both the detoxifying activities of Imuthiol and its influence on the T-cell lineage.
EXPERIMENTAL
PROCEDURES
Mice Female C3H/He mice were obtained from the Centre de Selection des Animaux de Laboratoire (CNRS, Orleans, France). They were maintained in an air-filtered and conditioned (24°C) room until use at 7 - 8 weeks of age, and fed with antibiotic-free commercial pellets and water ad libitum.
*Correspondence should be addressed to Dr. G6rard Renoux, 1 bis rue des Ursulines, 37000 Tours, France. 859
860
J.-M. GUILLAUMIN,A. LEPAPE and G. RENOUX
Reagents Imuthiol® (purified and lyophilised sodium diethyldithiocarbamate, Institut M6rieux, Lyon, France)was suspended in pyrogen-free saline together with diethyl-(35S)-dithiocarbamic acid, sodium salt (Amersham, Paris, France), so that 0.2 ml i.v. injected via the retroorbital route corresponded to 200 mg/kg body weight of Imuthiol and to 35/aCi labeled chromium (5~CrC12, spec. act. 500 mCi/mg, CEA, France) was adjusted so that 1 ml of the suspension contained 25/aCi: 0.2 ml were given i.v.
Radioactivity counting Prior to sacrifice, blood was withdrawn by retroorbital puncture under ether anesthesia, at the time intervals indicated in the Results. Plasma from three to five mice were pooled and recovered cell-free after spinning for 10 min at 4000 g. Liver, kidneys, spleen, thymus, axillary lymph nodes, white brain matter and right and left brain neocortex were rapidly sampled, defatted, and rinsed three times with cold saline to remove excess blood. To estimate the part of radioactive contamination by blood on 35S specific activity in tissues, organs were sampled as above after an i.v. injection of 51Cr for specific labeling of erythrocytes with 5tCr sodium chromate (Belcher, Berlin, Dudley, Garby, Heimpel, Lewis, Mcintyre, Mollison, Najean & Pettit, 1980). A weighed aliquot of each sample was digested with 0.5 ml of Soluene350 (Packard) for 12 h at 37°C, in borosilicated scintillation vials. Decoloration was then performed by incubation for 45 min at 40°C in the presence of 0.2 ml of isopropanol and 0.2 ml of a 30070 hydrogen peroxide solution. Unisolve (Koch Light Laboratories), containing 207o acetic acid to prevent chemoluminescence, was used as liquid scintillator. 35S activity was measured in a Kontron SL 4 220 counter, quenching correction being performed by external standardization. A Packard gamma counter was used for 5'Cr counts that were performed directly on samples.
Autoradiography Mice were given ether anesthesia and quickly frozen in dry ice - acetone mixture for 30 s, then kept at - 2 0 ° C for a week before sectioning. A Leitz cryomicrotome was employed to make 5 tam sections of the entire mouse. Slices were removed with adhesive Scotch tape and freeze-dried for a week in a - 20°C air stream. Dry sections were then applied on a high resolution X-ray film (Definix medical,
Kodak) for several months at - 20°C. Photographic processing included development with D 19 B (Kodak) and fixation with Hypam (Ilford).
Analytical procedures Neocortical brain tissue was carefully dissected, then weighed and homogenized in a mixture of dimethylsulfoxide in 1070sodium chloride and 0.01 M ethylene diamine tetra-acetic acid (25/100 v/v), according to Faiman, Dodd, Nolan, Artman & Hanzlik (1977). 35S radio activity was evaluated, then lipid extraction was performed in pentane. HPLC analysis was run on a Model 5000 Isocratic Liquid Chromatograph (Varian, Palo Alto, CA) equipped with 10/am alkylphenyl M Bondapak column (30 cm x 3.9 mm I.D., Waters Ass.) and an electrochemical detector (Tacussel-Solea, Lyon, France) for improved sensitivity. The methyl ester was prepared by reacting dithiocarbamate with methyl iodide, according to the technique described by Faiman et al. (1977).
RESULTS
Evaluation of ~5S activity in plasma and tissues The distribution of radioactive Imuthiol in plasma, liver, spleen, kidney, small intestine, lung, lymph node, thymus, white brain matter, and left and right brain neocortex after the i.v. administration of 35S-imuthiol to mice, is shown in Fig. 1. A 15 rain half-life of the labeled compound in plasma can be calculated from the data. In organs where the vascular supply represents a large amount of the total weight, an evaluation of labeled sulfur uptake by tissues would be artifactually shifted. Therefore, the quantity of blood remaining in organs was evaluated by counts of 5tCr radioactivity and subtracted from the crude counts. The results thus corrected are presented in Fig. 1. Five minutes after 35S-Imuthiol administration radioactivity was found in all tissues tested, with the exception of lung and white brain matter. Liver and thymus were important sites of uptake for 35S, plateauing for the time of observation. Brain neocortex and lymph nodes exhibited a similar kinetics for the uptake of the 35S-label: the peak of labeled sulfur content was attained at 5 min, and bound specific radioactivity remained nearly constant by the 45 min of observation. Spleen, kidney and gut exhibited elimination curves which paralleled that of blood plasma, suggesting a feeble affinity of the tracer for these tissues.
Fate and Distribution of Sodium Diethyldithiocarbamate
861
Fig. 3(a,b). Silver grain autoradiographic images of the body of mice after treatment with 3sS-Imuthiol. The film (a) shows that 5 min after dosing, the walls of arteries (intercostal vessels) accumulate the label densely. Densest labeling occurred in liver, kidney and intestinal lumen. White brain matter, lung, or organ fat envelopes were not detectably labeled. (b) obtained 30 min after injecting ~sS-Imuthiol, confirms the data derived from liquid scintillation counting of dissected tissues or organs: the tracer has accumulated in brain neocortex, while white brain matter was free of radioactivity; liver, kidney and gut remained deeply stained; neither fat or lymph nodes were demonstrably stained. Unfortunately, the thymus area was not kept in this section.
862
J.-M. GUILLAUMIN, A. LEPAPE and G. RENOUX
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Fig. 3(c,d). Autoradiographs m a d e 1 h post-dosing (c) reveal that the thymic area accumulates the tracer, and that liver, kidney and feces were selectively dense. The brain neocortex showed a lower level of density, especially in the left area. L u n g and fatty organ envelopes were no more densely labeled t h a n the cardiac area. As shown in (d), obtained 24 h after 35S-Imuthiol administration, the t h y m u s area was markedly labeled as well as the submaxillary lymph nodes; the radioactive tracer was still present in liver, kidney and gut lumen.
863
Fate and Distribution of Sodium Diethyldithiocarbamate As depicted in Fig. 2, the yield of 3~S in pentane extracts o f brain neocortex tissue displayed a slight time-associated increase in right neocortex and a feeble decrease by time in left neocortex. The differences were significant at 45 min ( P = 0 . 0 5 ) . Lipid-free material from brain neocortical tissue exhibited a low radioactivity incorporation, and a fast clearance curve. The findings suggested that the labeled c o m p o u n d was mostly localized on neocortical tissue lipids, and that Imuthiol was transformed in a more stable and apolar component when in contact with brain neocortex. The pentane extracted radioactive fraction of neocortical tissue was therefore co-chromatographied (alkylphenyl H P L C chromatography) with the methyl ester of dithiocarbamic acid. Both retention speeds were similarly located at 3.25 min, while the retention time of diethyldithiocarbamate was 2.29 min, and that of carbon disulfide was 3.40 min. The data show that methyl ester, a main metabolite of sodium diethyldithiocarbamate (Gessner & Jakuboswski, 1972), was the component localized on brain neocortex tissue.
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Fig. 1. 35S-Imuthiol was administered to mice i.v., the animals sacrificed sequentially at the time intervals indicated, and 3sS determined in plasma . The relative blood content of the the marker (~Cr evaluation) was deduced to evaluate the 35S scintillation counts in liver A . . . . . . . . . A; spleen x ×; kidney x ........ ×; small gut [] . . . . •; lymph node l---g; thymus • . . . . . . . . . . . . • ; left brain neocortex © O; right brain neocortex _" 0 . Data from white brain matter and lung are not represented as they are constantly below the sensitivity of the test.
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Fig. 2. 35S content of the lipid (pentane extractable) fractions of the left O O or right $ • brain neocortex, and of the aqueous (lipid-free) phase -~ - + of these tissues, sampled at the time periods indicated after the i.v. administration of 3SS-labeled lmuthiol.
Organ distribution Autoradiography was performed on whole mice sacrificed 5 rain, 30 min, 1 h and 24 h, respectively, after the i.v. dosing. Figure 3a shows that the radioactive material was maximally present in liver, kidneys and intestinal lumen within 5 min. The gray background corresponds to radioactivity linked with blood. However, the distribution did not seem related to blood vascular supply as the cardiac area, the meningeal structures, and the bone marrow were poorly labeled. Blood vessel sections in the thoracic area (intercostal vessels) show an intense, circular labeling mostly located in the inner membrane. This aspect would suggest an early metabolism of Imuthiol was needed for its transfer. Neither lung nor fat envelope o f organs such as lymph nodes or kidneys were detectably labeled. Figure 3b depicts that after 30 rain the tracer accumulated in brain neocortex while white matter and medulla oblongata remained free of radioactivity. The distribution previously observed (liver, kidney, gut) seemed more deeply stained than at 5 rain. Neither fat nor lymph nodes were demonstrably radiolabeled. Autoradiographs of 1-h-injected mice reveal (Fig. 3c) that brain neocortex labeling was fading, particularly in the left neocortex, whereas binding o f the tracer was visible in the thymic area. Radiomarking was augmented in liver, in comparison with the 30 min picture. Lungs, as well as fatty areas and bone marrow, remained unlabeled. The intense radioactivity in cortical and medullar kidney, and in
864
J.-M. GUILLAUMIN, A. LEPAPE and G. RENOUX
feces demonstrates an intestinal and urinary passage of most 35S, 1 h post i.v. administration. The data suggest that a second-step elimination process was induced after a first early (5 rain) elimination. Figure 3d reveals that the radioactive tracer was still present in liver, kidney and guts 24 h after administration. Thymus was then intensively labeled, as well as submaxillary lymph nodes. It might be that 35S binding to thymus tissue became more noticeable at this stage of observation than at earlier times, because background radioactivity was diminished in adjacent structures. DISCUSSION
This study is the first to show a localization of 35Slabeled Imuthiol on thymus and brain neocortex. The thymus location was never looked for before the present study, although radioactive thiol binding was nearly as high in the thymus as in the liver, on an equivalent organ weight basis. We have demonstrated a specific and durable location of the tracer in the brain neocortex, without detectable radioactivity in the white matter both by dissecting the gray matter from the white brain matter prior to evaluating their 35S content, and by the use of autoradiography technique. Str6mme & Eldjarn (1966) and Faiman, Dodd & Hanzilk (1978) were unable to find any radioactive thiol in brain after l h. Conceivably, the differences are due to experimental design, as tests performed by these authors involved the whole brain while present data show that the 35S-label was not detectable in the white brain matter by radioactivity counts or autoradiography. Affinity binding to thymus, brain neocortex and liver is most likely to occur as radioactivity was plateauing in these tissues. Autoradiographs confirm that a specific binding of the "S-label in liver, thymus and brain neocortex might be assumed, as labeled sulfur was still detectable 24 h after dosing. A binding to tissues can also be deduced from the lack of radioactive thiol in supernatant of liver homogenate (Str6mme & Eldjarn, 1966). Peak levels of radioactivity in liver, thymus and brain neocortex appeared within 5 min after "S i.v. administration. The long term uptake in thymus, liver and brain neocortex contrasts with the fast elimination rate in blood, spleen, kidney and gut, and also with the striking lack of lung radioactivity. The presence of labeled thiol in lung, kidney and gut have, however, been described (Str~Jmme & Eldjarn, 1966; Faiman et al., 1978). This appears to be
inconsistent with our finding. The difference is likely due to experimental design as blood radioactivity was deduced in our study, but not in earlier studies (Str6mme & Eldjarn, 1966; Faiman et al., 1978). Elimination through urine and feces of most "Slabel starts within minutes after dosing. It might correspond to a rapid metabolism and excretion of non-fixed Imuthiol. It is consistent with the finding of Gessner & Jakubowski (1972) and of Faiman et al. (1978) that dithiocarbamate was excreted by kidney and gut in the form of inorganic sulfate and glucuronide as early as 5 min after administration. Elimination via the lung of the 35S-label in the form of carbon disulfide has been documented in rats (Craven, Luscombe & Nicholls, 1976). This suggests that those organs which insure excretion are passage organs where Imuthiol is rapidly metabolized without binding. The rapid disappearance of imuthiol from blood is the result of tissue distribution and disulfide reduction. The lack of detectable radioactive material in organ envelope fat is an intriguing feature, as diethyldithiocarbamate and diethyldithiocarbamate methyl ester were described as lipophilic agents (Thorn & Ludwig, 1962). The ~-~S-labelwas identified as diethyldithiocarbamate-methyl ester in the pentane extractable brain neocortex lipids within 5 min post administration. This is consistent with the presence of diethyldithiocarbamate in the aqueous phase and of its methyl ester in the organic phase of tissue pentane extraction (Faiman et al., 1977). The data confirm that an extensive and rapid metabolism (Gessner & Jakubowski, 1972; Faiman et al., 1978) might be needed for binding and subsequent activity of imuthiol, through a specific enzymatic capacity present both in liver and brain (Feuer, Sosa-Lucero, Lumb & Moddel, 1971). The "S-label does not localize on lymphoid organs (spleen, bone marrow), strongly suggesting an indirect mode of action of Imuthiol was involved, as already demonstrated experimentally (Renoux, 1982; Pompidou et al., 1984; Renoux & Renoux, 1984; Pompidou, Duchet, Cooper, Mac6, Telvi, Coutance, Hadden & Renoux, 1985). The present data, showing that some type of concentration takes place in vivo provide a better understanding of the management of Imuthiol by the host at organ levels, and suggest the compound should be metabolized for immunostimulant activity. The organs (liver, thymus, brain neocortex) where the 35S-label binds correspond to those organs or tissues already shown as active sites by biological studies.
Fate and Distribution of Sodium Diethyldithiocarbamate
865
REFERENCES
BELCHER, E. H., BERLIN, N. I., DUDLEY, R. A., GARBY, L., HEIMPEL, H., LEWIS, S. M., MCINTYRE, P. A., MOLLISON,P. L., NAJEAN, Y. • PETTIT,J. E. (1980). Recommended methods for measurement of red cell and plasma volume. International Committee for Standardization in Haematology. J. nucl. Med., 21, 793- 800. CRAVEN, M. R., LUSCOMBE, D. K. & NICHOLLS, P. J. (1976). Absorption, elimination and duration of action of diethyldithiocarbamate in animals. J. pharm. Pharmac., 28, 38. FAIMAN, M. D., DODD, D. E. & HANZLIK, R. E. (1978). Distribution of S" disulfiram and metabolism of S" disulfiram in the dog. Res. Commun. Chem. Path. Pharmac., 21, 543-567. FAIMAN, M. D., DODD, D. E., NOLAN, R. J., ARTMAN,L. & HANZLIK,R. E. (1977). A rapid and simple radioactive method for the determination of disulfiram and its metabolites from a single sample of biological fluid or tissue. Res. Commun. Chem. Path. Pharmac., 17, 579-589. FEUER, G., SOSA-LUCERO,J. C., LUMB, G. 8,: MODDEL, G. (1971). Failure of various drugs to induce drug-metabolizing enzymes in extrahepatic tissues of the rat. Toxic. Appl. Pharmac., 19, 579- 589. GESSNER, T. & JAr~UBOWSKI,M. (1972). Diethyldithiocarbamic acid-methyl ester: a metabolite of disulfiram. Biochem. Pharmac., 21, 219-230. POMPIDOU, m., DUCHET, N., COOPER, M. D., MACle,B., TELVI, L., COUTANCE,F., HADDEN, J. W. & RENOUX,G. (1985). The generation and regulation of human lymphocytes by imuthiol" evidence from an in vitro differentiation induction system. Int. J. Immunopharmac., 7, 561- 566. POMP1DOU, A., RENOUX,M., GUILLAUMIN,J. M., MACI~,B., MICHEL, P., COUTANCE,F. & RENOUX,G. (1984). Kinetics of the histological changes in lymphoid organs and the T-cell inducing capacity of serum from mice treated with imuthiol (sodium diethyldithiocarbamate). Int. Archs Allergy appL Immun., 74, 172- 177. RENOUX, G. (1982). lmmunopharmacologie et pharmacologie du diethyldithiocarbamate de sodium (DTC). J. Pharmac., Paris, 13, 9 5 - 134. RENOUX, G. (1984). The mode of action of imuthiol (sodium diethyldithiocarbamate): a new role for the brain neocortex and the endocrine liver in the regulation of the T-cell lineage. In Immune Modulation Agents and their Mechanisms (eds Fenichel, R. L. and Chirigos, M. A.), pp. 607-624, Marcel Dekker, New York. RENOUX, G., BIZU~RE,K., RENOUX,M. & GUILLAUMIN,J. M. (1983). The production of T-cell inducing factors in mice is controlled by the brain neocortex. Scand. J. Immun., 17, 4 5 - 50. RENOUX, M., GIROUD, J. P., FLORENTIN, I., GUILLAUMIN,J. M., DEGENNE, D. & RENOUX, G. (1986). Early changes in immune parameters induced by an acute non antigenic inflammation in the mouse. Influence of Imuthiol. Int. J. Immunopharmac., 8, 107 - 117. RENOUX, G. & RENOUX, M. (1979). Immunopotentiation and anabolism induced by sodium diethyldithiocarbamate. J. Immunopharmac., 1, 247-267. RENOUX, G. & RENOUX,M. (1980). Administration of DTC. Evidence of a role of the thymus in the control and regulation of factors inducing thymocyte differentiation in the mouse. Thymus, 2, 139- 145. RENOUX, G. & RENOUX, M. (1983). DTC, a T-cell specific agent in nu/nu mice. In Current Drugs andMethods o f Cancer Treatment (eds Math6, G., Mihich, E. and Reizenstein, P.) pp. 171 - 173, Masson, New York. RENOUX, G., RENOUX, M., BIZII~RE, K., GUILLAUMIN,J. M., BARDOS, P. & DEGENNE, D. (1984). Involvement of brain neocortex and liver in the regulation of T cells: the mode of action of sodium diethyldithiocarbamate (imuthiol). Immunopharmacology, 7, 8 9 - 100. RENOUX, G., RENOUX, M. & GUILLAUMIN,J. M. (1982). Hepatosin. Int. J. Immunopharmac., 4, 300. RENOUX, G., TOURAINE, J. L. & RENOUX,M. (1980). Induction of differentiation of human null cells into T lymphocytes by the serum of mice treated with sodium diethyldithiocarbamate. J. lmmunopharmac., 2, 4 9 - 59. STROMME,J. H. ~,~ELDJARN,t . (1966). Distribution and chemical forms of diethyldithiocarbamate and tetraethylthiuram disulphide (disulfiram) in mice in relation to radioprotection. Biochem. Pharmac., 15, 287- 297. THORN, G. D. & LUDWIC, R. A. (1962). The Dithiocarbamates and Related Compounds. Elsevier, Amsterdam.
0192 0561/86 $3.00+ .00 International Society for lmmunopharmacology.
FATE AND DISTRIBUTION OF RADIOACTIVE SODIUM DIETHYLDITHIOCARBAMATE (IMUTHIOL ®) IN THE MOUSE JEAN-MAURICE GUILLAUMIN,ALAIN LEPAPE and GI~RARDRENOUX* Laboratoire d'Immunologie, Facult6 de M6decine, 37032 Tours Cedex, France (Received 17 October 1985 and in finalform 26 February 1986)
- - The distribution of 35S in mouse tissues has been investigated by radioactivity counts and by autoradiography after the intravenous injection of 35S-labeledimuthiol (sodium diethyldithiocarbamate). Radioactive Imuthiol is selectivelylocalized on liver, thymus and brain neocortex, most likely as the methyl ester, within minutes after dosing. Lung or white brain matter did not fix the labeled thiol. Blood, kidney and guts show rapid elimination patterns, and can be considered as passage organs which did not fix lmuthiol. The findings are consistent with the immunopharmacological data which demonstrate that Imuthiol exerts its T-cell recruiting and activating influence through a multi-step pathway involving the brain neocortex, the thymus and the liver.
Abstract
As an immunotherapeutic agent, Imuthiol® (purified sodium diethyldithiocarbamate), specifically recruits and activates T cells, as well as NK activity, and does not directly affect B cells and macrophages (Renoux & Renoux, 1979, 1980, 1983; Renoux, 1982; Pompidou, Renoux, Guillaumin, Mac6, Michel, Coutance & Renoux, 1984). Imuthiol can replace the inductive signals emitted by the left brain neocortex and it controls the synthesis of a T-cell specific liver factor, hepatosin (Renoux, Bizi6re, Renoux & Guillaumin, 1983; Renoux, Renoux, Bizi6re, Guillaumin, Bardos & Degenne, 1984). It increases the in vitro liver synthesis of hepatosin (Renoux, Renoux & Guillaumin, 1982; Renoux, 1984). The biological attributes of Imuthiol have been recently reviewed (Renoux, 1982). These include: destruction of neoplastic cells, parasites, fungi and bacteria at doses inactive on normal mammalian cells; a detoxifying influence against chemical carcinogens or agents toxic for the liver; an antistress effect through the regulation of dopamine/3 hydroxylase; an anti-inflammatory activity suggested by its influence on superoxide dismutase levels was recently confirmed (Renoux, Giroud, Florentin, Guillaumin, Degenne & Renoux, 1986). All these activities were thought to be associated with the
chelating and lipophilic properties of sodium diethyldithiocarbamate. A study was undertaken to reevaluate the distribution of the agent in the organs of mice by means of 35S-labeled Imuthiol in an attempt for a better understanding of the apparently complex mode of action of the agent. The data reported herein give evidence for a preferential localization of 35S on liver, thymus and brain neocortex. The findings fit in with the conclusions of the biological studies, which suggest that these three organs are involved in the multi-step pathway leading to both the detoxifying activities of Imuthiol and its influence on the T-cell lineage.
EXPERIMENTAL
PROCEDURES
Mice Female C3H/He mice were obtained from the Centre de Selection des Animaux de Laboratoire (CNRS, Orleans, France). They were maintained in an air-filtered and conditioned (24°C) room until use at 7 - 8 weeks of age, and fed with antibiotic-free commercial pellets and water ad libitum.
*Correspondence should be addressed to Dr. G6rard Renoux, 1 bis rue des Ursulines, 37000 Tours, France. 859
860
J.-M. GUILLAUMIN,A. LEPAPE and G. RENOUX
Reagents Imuthiol® (purified and lyophilised sodium diethyldithiocarbamate, Institut M6rieux, Lyon, France)was suspended in pyrogen-free saline together with diethyl-(35S)-dithiocarbamic acid, sodium salt (Amersham, Paris, France), so that 0.2 ml i.v. injected via the retroorbital route corresponded to 200 mg/kg body weight of Imuthiol and to 35/aCi labeled chromium (5~CrC12, spec. act. 500 mCi/mg, CEA, France) was adjusted so that 1 ml of the suspension contained 25/aCi: 0.2 ml were given i.v.
Radioactivity counting Prior to sacrifice, blood was withdrawn by retroorbital puncture under ether anesthesia, at the time intervals indicated in the Results. Plasma from three to five mice were pooled and recovered cell-free after spinning for 10 min at 4000 g. Liver, kidneys, spleen, thymus, axillary lymph nodes, white brain matter and right and left brain neocortex were rapidly sampled, defatted, and rinsed three times with cold saline to remove excess blood. To estimate the part of radioactive contamination by blood on 35S specific activity in tissues, organs were sampled as above after an i.v. injection of 51Cr for specific labeling of erythrocytes with 5tCr sodium chromate (Belcher, Berlin, Dudley, Garby, Heimpel, Lewis, Mcintyre, Mollison, Najean & Pettit, 1980). A weighed aliquot of each sample was digested with 0.5 ml of Soluene350 (Packard) for 12 h at 37°C, in borosilicated scintillation vials. Decoloration was then performed by incubation for 45 min at 40°C in the presence of 0.2 ml of isopropanol and 0.2 ml of a 30070 hydrogen peroxide solution. Unisolve (Koch Light Laboratories), containing 207o acetic acid to prevent chemoluminescence, was used as liquid scintillator. 35S activity was measured in a Kontron SL 4 220 counter, quenching correction being performed by external standardization. A Packard gamma counter was used for 5'Cr counts that were performed directly on samples.
Autoradiography Mice were given ether anesthesia and quickly frozen in dry ice - acetone mixture for 30 s, then kept at - 2 0 ° C for a week before sectioning. A Leitz cryomicrotome was employed to make 5 tam sections of the entire mouse. Slices were removed with adhesive Scotch tape and freeze-dried for a week in a - 20°C air stream. Dry sections were then applied on a high resolution X-ray film (Definix medical,
Kodak) for several months at - 20°C. Photographic processing included development with D 19 B (Kodak) and fixation with Hypam (Ilford).
Analytical procedures Neocortical brain tissue was carefully dissected, then weighed and homogenized in a mixture of dimethylsulfoxide in 1070sodium chloride and 0.01 M ethylene diamine tetra-acetic acid (25/100 v/v), according to Faiman, Dodd, Nolan, Artman & Hanzlik (1977). 35S radio activity was evaluated, then lipid extraction was performed in pentane. HPLC analysis was run on a Model 5000 Isocratic Liquid Chromatograph (Varian, Palo Alto, CA) equipped with 10/am alkylphenyl M Bondapak column (30 cm x 3.9 mm I.D., Waters Ass.) and an electrochemical detector (Tacussel-Solea, Lyon, France) for improved sensitivity. The methyl ester was prepared by reacting dithiocarbamate with methyl iodide, according to the technique described by Faiman et al. (1977).
RESULTS
Evaluation of ~5S activity in plasma and tissues The distribution of radioactive Imuthiol in plasma, liver, spleen, kidney, small intestine, lung, lymph node, thymus, white brain matter, and left and right brain neocortex after the i.v. administration of 35S-imuthiol to mice, is shown in Fig. 1. A 15 rain half-life of the labeled compound in plasma can be calculated from the data. In organs where the vascular supply represents a large amount of the total weight, an evaluation of labeled sulfur uptake by tissues would be artifactually shifted. Therefore, the quantity of blood remaining in organs was evaluated by counts of 5tCr radioactivity and subtracted from the crude counts. The results thus corrected are presented in Fig. 1. Five minutes after 35S-Imuthiol administration radioactivity was found in all tissues tested, with the exception of lung and white brain matter. Liver and thymus were important sites of uptake for 35S, plateauing for the time of observation. Brain neocortex and lymph nodes exhibited a similar kinetics for the uptake of the 35S-label: the peak of labeled sulfur content was attained at 5 min, and bound specific radioactivity remained nearly constant by the 45 min of observation. Spleen, kidney and gut exhibited elimination curves which paralleled that of blood plasma, suggesting a feeble affinity of the tracer for these tissues.
Fate and Distribution of Sodium Diethyldithiocarbamate
861
Fig. 3(a,b). Silver grain autoradiographic images of the body of mice after treatment with 3sS-Imuthiol. The film (a) shows that 5 min after dosing, the walls of arteries (intercostal vessels) accumulate the label densely. Densest labeling occurred in liver, kidney and intestinal lumen. White brain matter, lung, or organ fat envelopes were not detectably labeled. (b) obtained 30 min after injecting ~sS-Imuthiol, confirms the data derived from liquid scintillation counting of dissected tissues or organs: the tracer has accumulated in brain neocortex, while white brain matter was free of radioactivity; liver, kidney and gut remained deeply stained; neither fat or lymph nodes were demonstrably stained. Unfortunately, the thymus area was not kept in this section.
862
J.-M. GUILLAUMIN, A. LEPAPE and G. RENOUX
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Fig. 3(c,d). Autoradiographs m a d e 1 h post-dosing (c) reveal that the thymic area accumulates the tracer, and that liver, kidney and feces were selectively dense. The brain neocortex showed a lower level of density, especially in the left area. L u n g and fatty organ envelopes were no more densely labeled t h a n the cardiac area. As shown in (d), obtained 24 h after 35S-Imuthiol administration, the t h y m u s area was markedly labeled as well as the submaxillary lymph nodes; the radioactive tracer was still present in liver, kidney and gut lumen.
863
Fate and Distribution of Sodium Diethyldithiocarbamate As depicted in Fig. 2, the yield of 3~S in pentane extracts o f brain neocortex tissue displayed a slight time-associated increase in right neocortex and a feeble decrease by time in left neocortex. The differences were significant at 45 min ( P = 0 . 0 5 ) . Lipid-free material from brain neocortical tissue exhibited a low radioactivity incorporation, and a fast clearance curve. The findings suggested that the labeled c o m p o u n d was mostly localized on neocortical tissue lipids, and that Imuthiol was transformed in a more stable and apolar component when in contact with brain neocortex. The pentane extracted radioactive fraction of neocortical tissue was therefore co-chromatographied (alkylphenyl H P L C chromatography) with the methyl ester of dithiocarbamic acid. Both retention speeds were similarly located at 3.25 min, while the retention time of diethyldithiocarbamate was 2.29 min, and that of carbon disulfide was 3.40 min. The data show that methyl ester, a main metabolite of sodium diethyldithiocarbamate (Gessner & Jakuboswski, 1972), was the component localized on brain neocortex tissue.
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Fig. 1. 35S-Imuthiol was administered to mice i.v., the animals sacrificed sequentially at the time intervals indicated, and 3sS determined in plasma . The relative blood content of the the marker (~Cr evaluation) was deduced to evaluate the 35S scintillation counts in liver A . . . . . . . . . A; spleen x ×; kidney x ........ ×; small gut [] . . . . •; lymph node l---g; thymus • . . . . . . . . . . . . • ; left brain neocortex © O; right brain neocortex _" 0 . Data from white brain matter and lung are not represented as they are constantly below the sensitivity of the test.
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Fig. 2. 35S content of the lipid (pentane extractable) fractions of the left O O or right $ • brain neocortex, and of the aqueous (lipid-free) phase -~ - + of these tissues, sampled at the time periods indicated after the i.v. administration of 3SS-labeled lmuthiol.
Organ distribution Autoradiography was performed on whole mice sacrificed 5 rain, 30 min, 1 h and 24 h, respectively, after the i.v. dosing. Figure 3a shows that the radioactive material was maximally present in liver, kidneys and intestinal lumen within 5 min. The gray background corresponds to radioactivity linked with blood. However, the distribution did not seem related to blood vascular supply as the cardiac area, the meningeal structures, and the bone marrow were poorly labeled. Blood vessel sections in the thoracic area (intercostal vessels) show an intense, circular labeling mostly located in the inner membrane. This aspect would suggest an early metabolism of Imuthiol was needed for its transfer. Neither lung nor fat envelope o f organs such as lymph nodes or kidneys were detectably labeled. Figure 3b depicts that after 30 rain the tracer accumulated in brain neocortex while white matter and medulla oblongata remained free of radioactivity. The distribution previously observed (liver, kidney, gut) seemed more deeply stained than at 5 rain. Neither fat nor lymph nodes were demonstrably radiolabeled. Autoradiographs of 1-h-injected mice reveal (Fig. 3c) that brain neocortex labeling was fading, particularly in the left neocortex, whereas binding o f the tracer was visible in the thymic area. Radiomarking was augmented in liver, in comparison with the 30 min picture. Lungs, as well as fatty areas and bone marrow, remained unlabeled. The intense radioactivity in cortical and medullar kidney, and in
864
J.-M. GUILLAUMIN, A. LEPAPE and G. RENOUX
feces demonstrates an intestinal and urinary passage of most 35S, 1 h post i.v. administration. The data suggest that a second-step elimination process was induced after a first early (5 rain) elimination. Figure 3d reveals that the radioactive tracer was still present in liver, kidney and guts 24 h after administration. Thymus was then intensively labeled, as well as submaxillary lymph nodes. It might be that 35S binding to thymus tissue became more noticeable at this stage of observation than at earlier times, because background radioactivity was diminished in adjacent structures. DISCUSSION
This study is the first to show a localization of 35Slabeled Imuthiol on thymus and brain neocortex. The thymus location was never looked for before the present study, although radioactive thiol binding was nearly as high in the thymus as in the liver, on an equivalent organ weight basis. We have demonstrated a specific and durable location of the tracer in the brain neocortex, without detectable radioactivity in the white matter both by dissecting the gray matter from the white brain matter prior to evaluating their 35S content, and by the use of autoradiography technique. Str6mme & Eldjarn (1966) and Faiman, Dodd & Hanzilk (1978) were unable to find any radioactive thiol in brain after l h. Conceivably, the differences are due to experimental design, as tests performed by these authors involved the whole brain while present data show that the 35S-label was not detectable in the white brain matter by radioactivity counts or autoradiography. Affinity binding to thymus, brain neocortex and liver is most likely to occur as radioactivity was plateauing in these tissues. Autoradiographs confirm that a specific binding of the "S-label in liver, thymus and brain neocortex might be assumed, as labeled sulfur was still detectable 24 h after dosing. A binding to tissues can also be deduced from the lack of radioactive thiol in supernatant of liver homogenate (Str6mme & Eldjarn, 1966). Peak levels of radioactivity in liver, thymus and brain neocortex appeared within 5 min after "S i.v. administration. The long term uptake in thymus, liver and brain neocortex contrasts with the fast elimination rate in blood, spleen, kidney and gut, and also with the striking lack of lung radioactivity. The presence of labeled thiol in lung, kidney and gut have, however, been described (Str~Jmme & Eldjarn, 1966; Faiman et al., 1978). This appears to be
inconsistent with our finding. The difference is likely due to experimental design as blood radioactivity was deduced in our study, but not in earlier studies (Str6mme & Eldjarn, 1966; Faiman et al., 1978). Elimination through urine and feces of most "Slabel starts within minutes after dosing. It might correspond to a rapid metabolism and excretion of non-fixed Imuthiol. It is consistent with the finding of Gessner & Jakubowski (1972) and of Faiman et al. (1978) that dithiocarbamate was excreted by kidney and gut in the form of inorganic sulfate and glucuronide as early as 5 min after administration. Elimination via the lung of the 35S-label in the form of carbon disulfide has been documented in rats (Craven, Luscombe & Nicholls, 1976). This suggests that those organs which insure excretion are passage organs where Imuthiol is rapidly metabolized without binding. The rapid disappearance of imuthiol from blood is the result of tissue distribution and disulfide reduction. The lack of detectable radioactive material in organ envelope fat is an intriguing feature, as diethyldithiocarbamate and diethyldithiocarbamate methyl ester were described as lipophilic agents (Thorn & Ludwig, 1962). The ~-~S-labelwas identified as diethyldithiocarbamate-methyl ester in the pentane extractable brain neocortex lipids within 5 min post administration. This is consistent with the presence of diethyldithiocarbamate in the aqueous phase and of its methyl ester in the organic phase of tissue pentane extraction (Faiman et al., 1977). The data confirm that an extensive and rapid metabolism (Gessner & Jakubowski, 1972; Faiman et al., 1978) might be needed for binding and subsequent activity of imuthiol, through a specific enzymatic capacity present both in liver and brain (Feuer, Sosa-Lucero, Lumb & Moddel, 1971). The "S-label does not localize on lymphoid organs (spleen, bone marrow), strongly suggesting an indirect mode of action of Imuthiol was involved, as already demonstrated experimentally (Renoux, 1982; Pompidou et al., 1984; Renoux & Renoux, 1984; Pompidou, Duchet, Cooper, Mac6, Telvi, Coutance, Hadden & Renoux, 1985). The present data, showing that some type of concentration takes place in vivo provide a better understanding of the management of Imuthiol by the host at organ levels, and suggest the compound should be metabolized for immunostimulant activity. The organs (liver, thymus, brain neocortex) where the 35S-label binds correspond to those organs or tissues already shown as active sites by biological studies.
Fate and Distribution of Sodium Diethyldithiocarbamate
865
REFERENCES
BELCHER, E. H., BERLIN, N. I., DUDLEY, R. A., GARBY, L., HEIMPEL, H., LEWIS, S. M., MCINTYRE, P. A., MOLLISON,P. L., NAJEAN, Y. • PETTIT,J. E. (1980). Recommended methods for measurement of red cell and plasma volume. International Committee for Standardization in Haematology. J. nucl. Med., 21, 793- 800. CRAVEN, M. R., LUSCOMBE, D. K. & NICHOLLS, P. J. (1976). Absorption, elimination and duration of action of diethyldithiocarbamate in animals. J. pharm. Pharmac., 28, 38. FAIMAN, M. D., DODD, D. E. & HANZLIK, R. E. (1978). Distribution of S" disulfiram and metabolism of S" disulfiram in the dog. Res. Commun. Chem. Path. Pharmac., 21, 543-567. FAIMAN, M. D., DODD, D. E., NOLAN, R. J., ARTMAN,L. & HANZLIK,R. E. (1977). A rapid and simple radioactive method for the determination of disulfiram and its metabolites from a single sample of biological fluid or tissue. Res. Commun. Chem. Path. Pharmac., 17, 579-589. FEUER, G., SOSA-LUCERO,J. C., LUMB, G. 8,: MODDEL, G. (1971). Failure of various drugs to induce drug-metabolizing enzymes in extrahepatic tissues of the rat. Toxic. Appl. Pharmac., 19, 579- 589. GESSNER, T. & JAr~UBOWSKI,M. (1972). Diethyldithiocarbamic acid-methyl ester: a metabolite of disulfiram. Biochem. Pharmac., 21, 219-230. POMPIDOU, m., DUCHET, N., COOPER, M. D., MACle,B., TELVI, L., COUTANCE,F., HADDEN, J. W. & RENOUX,G. (1985). The generation and regulation of human lymphocytes by imuthiol" evidence from an in vitro differentiation induction system. Int. J. Immunopharmac., 7, 561- 566. POMP1DOU, A., RENOUX,M., GUILLAUMIN,J. M., MACI~,B., MICHEL, P., COUTANCE,F. & RENOUX,G. (1984). Kinetics of the histological changes in lymphoid organs and the T-cell inducing capacity of serum from mice treated with imuthiol (sodium diethyldithiocarbamate). Int. Archs Allergy appL Immun., 74, 172- 177. RENOUX, G. (1982). lmmunopharmacologie et pharmacologie du diethyldithiocarbamate de sodium (DTC). J. Pharmac., Paris, 13, 9 5 - 134. RENOUX, G. (1984). The mode of action of imuthiol (sodium diethyldithiocarbamate): a new role for the brain neocortex and the endocrine liver in the regulation of the T-cell lineage. In Immune Modulation Agents and their Mechanisms (eds Fenichel, R. L. and Chirigos, M. A.), pp. 607-624, Marcel Dekker, New York. RENOUX, G., BIZU~RE,K., RENOUX,M. & GUILLAUMIN,J. M. (1983). The production of T-cell inducing factors in mice is controlled by the brain neocortex. Scand. J. Immun., 17, 4 5 - 50. RENOUX, M., GIROUD, J. P., FLORENTIN, I., GUILLAUMIN,J. M., DEGENNE, D. & RENOUX, G. (1986). Early changes in immune parameters induced by an acute non antigenic inflammation in the mouse. Influence of Imuthiol. Int. J. Immunopharmac., 8, 107 - 117. RENOUX, G. & RENOUX, M. (1979). Immunopotentiation and anabolism induced by sodium diethyldithiocarbamate. J. Immunopharmac., 1, 247-267. RENOUX, G. & RENOUX,M. (1980). Administration of DTC. Evidence of a role of the thymus in the control and regulation of factors inducing thymocyte differentiation in the mouse. Thymus, 2, 139- 145. RENOUX, G. & RENOUX, M. (1983). DTC, a T-cell specific agent in nu/nu mice. In Current Drugs andMethods o f Cancer Treatment (eds Math6, G., Mihich, E. and Reizenstein, P.) pp. 171 - 173, Masson, New York. RENOUX, G., RENOUX, M., BIZII~RE, K., GUILLAUMIN,J. M., BARDOS, P. & DEGENNE, D. (1984). Involvement of brain neocortex and liver in the regulation of T cells: the mode of action of sodium diethyldithiocarbamate (imuthiol). Immunopharmacology, 7, 8 9 - 100. RENOUX, G., RENOUX, M. & GUILLAUMIN,J. M. (1982). Hepatosin. Int. J. Immunopharmac., 4, 300. RENOUX, G., TOURAINE, J. L. & RENOUX,M. (1980). Induction of differentiation of human null cells into T lymphocytes by the serum of mice treated with sodium diethyldithiocarbamate. J. lmmunopharmac., 2, 4 9 - 59. STROMME,J. H. ~,~ELDJARN,t . (1966). Distribution and chemical forms of diethyldithiocarbamate and tetraethylthiuram disulphide (disulfiram) in mice in relation to radioprotection. Biochem. Pharmac., 15, 287- 297. THORN, G. D. & LUDWIC, R. A. (1962). The Dithiocarbamates and Related Compounds. Elsevier, Amsterdam.