Comparative Toxicities of Mimosine and Some Chemically Related Compounds to Mouse Bone Marrow Cells in Liquid Culture
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Aust. J. Bioi. Sci., 1978, 31, 115-21 Comparative Toxicities of Mimosine and Some Chemically Related Compounds to Mouse Bone Marrow Cells in Liquid Culture M. P. Hegarty/ C. P. Lee,B G. S. Christie,B F. G. De MunkB and R. D. CourtA A Division of Tropical Crops and Pastures, CSIRO, Cunningham Laboratory, Mill Road, St Lucia, Qld 4067. B Pathology Department, University of Melbourne, Parkville, Vic. 3052. Abstract Mimosine, a plant amino acid which is toxic in mammals, was shown to be a potent inhibitor of incorporation of [3H)thymidine in mouse bone marrow cells in liquid culture (> 70 % inhibition at a concentration of 2 x 10-4 M). To determine the parts of the molecule responsible for the inhibitory mechanism the effects of 13 chemically related compounds were examined in this system. The structural features necessary for inhibitory activity of the 4(IH)-pyridones were (1) the 3-hydroxyl-4-oxo function of the pyridone ring together with (2) an IX-alanine or a 2-aminoethyl side chain. Compounds based on several other hydroxy heterocyclic functions were either weakly active or inactive. 3-Hydroxy-4(IH)-pyridone, the goitrogen to which mimosine is converted in ruminants, was only slightly inhibitory. These results are compared with published information on the effects of some of these compounds on other types of mammalian cells in vitro. The mouse bone marrow system in which inhibition of incorporation of [3Hlthymidine is used as an index of cytotoxicity was shown to be sensitive and reproducible, and could be useful for structure-activity investigations of other cytotoxic compounds. Introduction Mimosine is a non-protein amino acid which occurs in Mimosa pudica, and in large amounts (e.g. 8-10% of the dry weight of the growing tip) in Leucaena leuco cephala, a leguminous shrub which is finding increasing use as a pasture legume in sub-tropical Australia. Mimosine produces a variety of physiological effects when ingested by animals (for reviews see Owen 1958; Thompson et af. 1969; Hegarty and Peterson 1973). Mimosine has been found to inhibit certain kinds of tumours (De Wys and Hall 1973; Prabhakaran et al. 1973) and is therefore potentially useful as a model com pound in the study of these tumours. Its potential use as a chemical defleecing agent in sheep is being investigated in detail (Reis et al. 1975a, 1975b). 3-Hydroxy-4(1H) pyridone (DHP), to which mimosine is converted in the rumen of cattle, sheep and goats, has been shown to be a potent goitrogen although mimosine itself is not goitrogenic (Hegarty et al. 1976). These recent results emphasize the need for more detailed information on the biological properties of mimosine and related compounds so that compounds with enhanced activity may be prepared, and measures to overcome toxicity may be devised. The toxicity of mimosine to rapidly growing cells in vivo indicated that cultures of such cells should be suitable for studying the growth inhibitory properties in vitro. Tsai and Ling (1971, 1972), using H.Ep-2 cells grown as a monolayer, showed that mimosine inhibited [3H]thymidine incorporation and that DHP was less active than 116 M. P. Hegartyet al. mimosine. Ward and Harris (1976) showed that mimosine (2 x 10-4 M) was a potent inhibitor of the incorporation of [3H]thymidine by sheep skin slices in vitro and studied the inhibitory properties of 14 related 4(lH)-pyridones in this system. We have independently investigated the inhibitory properties of mimosine and 13 related compounds in a cell culture system. The compounds were selected so that the contributions made by the main structural features of the mimosine molecule to its biological activity could be tested. A culture system of mouse bone marrow cells was chosen because: (1) the cells can be freshly prepared from normal tissue for each experiment; (2) the cells are mesenchymal, and therefore representative of an important cell population hitherto untested, as previous workers used epithelial lines (epidermoid carcinoma and skin); and (3) the ability of the cells to grow as a sus pension in a liquid medium allows rapid and accurate volumetric control of the cell numbers inoculated into the replicate culture tubes, and uniform access of nutrients and reagents. These factors contribute to the sensitivity and reproducibility of the system. Inhibition of incorporation of [3H]thymidine. is used as a convenient and precise index of cytotoxicity, and the system has been shown to be reliable in testing the growth inhibitory properties of another non-protein amino acid (De Munk et al. 1971, 1972). Materials and Methods Liquid Culture System Nucleated marrow cells were aspirated from the femoral shafts of male mice (C57 BL/6J strain) under aseptic conditions and suspended in the culture medium at a concentration of 5 x 105 cells/ml determined by counting in a haemocytometer chamber. Then 1 ml of the suspension was transferred to each culture tube, and the tubes loosely capped and gassed with 10% CO2 in air during incubation at 37°C in a humid incubator (De Munk: et al. 1971, 1972). Medium Eagle's minimum essential growth medium was supplemented with sodium pyruvate (2' 2 % w/v to give a final concentration of 0'5%), serine (0'4% w/v to give a final concentration of 0·2 IUM) and foetal calf serum (25 %). Pencillin (10000 units/I) and streptomycin sulphate (50 mg/I) were also added to the medium. The components of the medium were purchased from the Commonwealth Serum Laboratories, Melbourne. Colony Stimulating Activity Extract This was prepared by the method of Bradley et al. (1971) and added to all culture tubes (50 .ul/ml). [3 Hl Thymidine Uptake Assay The methods used to assay the uptake of [3Hlthymidine in this system have been described in detail previously (De Munk et al. 1971, 1972). Briefly, the method involved the addition of [3H]thymidine (1 .uCi in IO.ul) (Radiochemical Centre, Amersham) to each culture tube for a 4-h period. After 4 h, uptake of [3Hlthymidine was stopped by the addition of ice-cold saline and excess [3Hlthymidine was then removed by centrifugation and washing with ice-cold saline followed by ice-cold 5 % trichloroacetic acid. The acid-insoluble precipitate was dissolved in 1 M NaOH and the incorporated [3Hlthymidine determined by liquid scintillation spectrometry (De Munk et al. 1971). The counts were corrected for quenching by the addition of an internal standard. The time at which the 4-h pulse of [3Hlthymidine was added was determined for each batch of colony stimulating activity extract (see De Munk et al. 1971, fig. 1) and was chosen as the day before the time of maximum uptake of [lH]thymidine by the culture. For these experiments it was added on day 6. Experiments Preliminary experiments with cultures from male Balb/c mice as used by De Munk: et al. (1972) indicated that mimosine was a potent inhibitor (about 80% inhibition at 2x 10-4 M) of incorporation Toxicities of Mimosine and Related Compounds 117 of [3H]thymidine, and that the system was sufficiently sensitive for testing the inhibitory activity of chemically related substances. Cultures from C57 BL/6J mice were used in the experiments reported here because we had access to a continuing supply of these mice. The test compounds were added to culture tubes to finaI concentrations of 2x 10-6 , 2x 10-5, 2x 10-4 and 2x 10-3 M at the time of inoculation with cell suspension. The tubes were then incubated and the uptake of [3Hjthymidine measured as described above. 0 R4 £('I N I RZ 0 !l HOD Rl RZ R3 R4 I ° I CHzCHC02H (1) L-CH2CH(NHz)C02H H OH H I (2) DL-CH2CH(NH2)C02H H OH H NH2 (13) (3) L-CH2CH(NH2)C02CH3 H OH H (4) H H OH DL -CHzCH(NHz)COzH (5) H H OH H (6) DL-CH2CH(NH 2)C02H CH3 OH H ° (7) H H OCH3 H H0'CNH (8) -CH2CHzNH2 H OH H (9) -CH2CH2NHz H OCH3 H I NA CH2CHCOzH I (10) DL -CH2CH( OH)C02H H OH H NH2 (n) DL -CH2CH(NH2)C02H H H H (14) (12) -CH2CH(NHCOCH2NH2)C02H H OH H Fig. 1. Chemical structures of compounds tested for inhibitory effects on uptake of [3Hjthymidine in mouse bone marrow cells in vitro. L-Mimosine (1), DL-mimosine (2), L-mimosine methyl ester (3), DL-oc-amino-p-(5-hydroxy-4-oxo-2(4H)-pyridine)propionic acid ('isomimosine') (4), 3-hydroxy- 4(1H)-pyridone (5), DL-2-methylmimosine (6), 3-methoxy-4(1H)-pyridone (7), 1(2'-aminoethyl)- 3-hydroxy-4(1H)-pyridone ('mimosinamine') (8), 3-0-methylmimosinamine (9), DL-oc-hydroxy desaminomimosine (10), DL-3-deoxymimosine (11), glycylmimosine (12), DL-5-hydroxy-4-pyrone- 2-oc-aminopropionic acid (13), DL-oc-amino-p-(5-hydroxy-4-oxo-3,4-dihydropyrimidin-2-yl)propionic acid (14). Test Compounds L-Mimosine was isolated by the method of Hegarty et al. (1964), and from it the following substances were prepared: 3-hydroxy-4(1H)-pyridone (DHP) (5) as described by Hegarty et al. (1964); L-mimosine methyl ester (3) by a modification of the method of Adams et- al. (1945); DL-oc-hydroxy-desaminomimosine (10) by deamination by the technique of Witkop and Foltz (1957). Mimosinamine (8) and 3-0-methylmimosinamine (9) were synthesized as described by Takahara and Takahashi (1972), and 3-methoxy-4(1H)-pyridone (7) by the method of Spenser and Notation (1962). The following compounds were obtained from Dr R. Harris, Division of Plant Industry, CSIRO, and details of the synthesis of most of them have been published (Harris 1976a, 1976b): DL-mimosine (2), DL-isomimosine (4), DL-2-methylmimosine (6), DL-3-deoxymimosine (11), DL-OC amino-p-(5-hydroxy-4-oxo-3,4-dihydro-pyrimidin-2-yl)propionic acid (14), and DL-5-hydroxy-4- pyrone-2-oc-aminopropionic acid (13).