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 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 , 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 , 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 . 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 , sheep and , 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). Glycyl mimosine (12) was obtained from Dr F. H. Stewart, Division of Protein Chemistry, CSIRO. The purity of the compounds was tested by chromatography and high voltage electrophoresis on thin layers of cellulose, and the use of a range of detecting reagents (ninhydrin, ferric chloride and iodine vapour). Stock solutions of the substances were prepared in O· 9 % saline or, if necessary, in slightly alkaline isotonic saline. The solutions were sterilized by filtering through a MiIIipore filter (0'45 .urn) before being added to the culture tubes. 118 M. P. Hegarty et al.

Results and Discussion The effects of L-mimosine and 13 related substances on the uptake of [3H]thymidine by mouse bone marrow cells in liquid culture are shown in Table 1. The majority of compounds caused complete inhibition of uptake at a concentration of 2 x 10 - 3 M. We have classified the compounds as active, weakly active, or inactive, based on the degree of inhibition produced at a concentration of 2 x 10-4 M. Active compounds produced inhibition> 70 %(mean value) (Table la), weakly active < 70 %but> 30 % (Table Ib) and inactive <30% (Table Ie). Reis et al. (1975a) found that defieecing

Table 1. Effect of mimosine and related compounds on [3H]tbymidine incorporation into DNA by cultured mouse bone marrow cells Each value is a mean of three experiments in which five replicate cultures tubes were set up for each compound at each concentration and the corrected cpm values compared with those of five replicate control tubes. Details of the culture method are described in the text

Compound % Inhibition (±s.d.) relative to control value, at concentrations (M) of: 2x 10-6 2x 10-5 2x 10-4A 2x 10-3

(a) Active L-Mimosine (1) 3±4 16±7 75±10 l00±O DL-Mimosine (2) 10±4 41±12 80±10 l00±O Mimosinamine (8) 6±1 13±7 74±7 IOO±O L-Mimosine methyl ester (3) 6±9 27±I2 80±I3 IOO±O DL-2-MethylmimosineB (6) 3±3 6±4 93±3 IOO±O DL-Isomimosine (4) H2 10±4 92±1 IOO±O (b) Weakly active DL-5-Hydroxy-4-pyrone- 2-IX-aminopropionicacid (13) 5±8 IO±IO 48±I5 99±1 (c) Inactive 3-Hydroxy-4(IH)-pyridone (5) 5±3 4±3 I2±5 IOO±O 3-Methoxy-4(1H)-pyridone (7) HI 5±2 lO±I 56±13 3-0-Methylmimosinamine (9) HI 4±2 26±IO 83±4 DL-3-Deoxymimosine (11) HI 6±2 I3±4 37±4 DL-IX-Hydroxy-desaminomimosine (10) 0 0 0 97±I Glycylmimosine (12) HI 5 11±7 IOO±O DL-IX-Amino-f1-(5-hydroxy-4-oxo-3,4-dihydro- pyrimidin-2-yl) propionic acid (14) 7±3 I8H 28±5 73±I

A Compounds defined as active if inhibition at this concentration > 70 %; weakly active if ,,70 % but > 30 %, and inactive is < 30 %. B Two experiments each of five replicate tubes. ensued when the concentration of mimosine in the plasma was maintained above 1 x 10-4 M for at least 30 h. Mimosine and the other compounds listed in Table la were potent inhibitors in the system and caused a measurable inhibition at 2 x 10-5 M, a concentration lower than that of many of the endogenous amino acids in the medium. Racemization, esterification, decarboxylation or shifting of the side chain (as in compounds 2, 3, 8 and 4 respectively) had little effect on the inhibitory properties of the compounds. However, binding the amino group in peptide linkage (compound 12) or removal of it from the side chain leaving an anionic structure (compound 10) resulted in loss of activity. The 3-hydroxy group of the pyridine ring was essential Toxicities of Mimosine and Related Compounds 119

for the activity of the 3-hydroxy-4(IH)-pyridone series of compounds as these lost activity when the group was methylated (compound 9) or absent (compound 11). Compound 14, a pyrimidine analogue of isomimosine, was inactive in our system, whilst compound 13, which contains a 4-pyrone ring instead of a 4-pyridone ring but has other structural features resembling isomimosine, showed weak activity. Mimosine (at a concentration of 2 x 10-4 M) has now been shown to be a potent inhibitor of incorporation of [3H]thymidine in vitro in H.Ep-2 cells (Tsai and Ling 1971, 1972), sheep skin slices (Ward and Harris 1976) and mouse bone marrow cells. Not all the compounds we have tested have been investigated in the other systems but compounds 9 and 12 have been found to have low activity in the sheep skin slice system (K. A. Ward and R. L. N. Harris, personal communication).

Table 2. Comparison of inhibitory effects of mimosine, mimosinamine and 3-hydroxy-4(1H)-pyridone on incorporation of [3H]thymidine in three cell types in vitro n.t., Not tested

Substance % Inhibition of incorporation of [3H]thymidine at a concentration of 2 x 10-4 M by: Mouse bone Sheep skin H.Ep-2 marrow cells slicesA celIsB

Mimosine 75 72 77 Mimosinamine 74 26 n.t. 3-Hydroxy-4(IH)-pyridone 12 65 96c

A Data from Ward and Harris (1976). B Data from Tsai and Ling (1971). c Tested at a concentration of 4 x 10-4 M.

With two exceptions those compounds which we have classified as active from our experiments have been active in the system of Ward and Harris (1976). The exceptions are DHP (compound 5) and mimosinamine (compound 8). Table 2 shows a comparison of the inhibitory effects of compounds 1, 5 and 8 on incorporation of [3H]thymidine into DNA in mouse bone marrow cells with those reported for H.Ep-2 cells (Tsai and Ling 1971) and sheep skin slices (Ward and Harris 1976). The discrepancies may be due to the use of different cell types as the test systems. Both H.Ep-2 (human epidermoid carcinoma) cells and the active cells in the sheep skin slices are epidermoid, whilst the cells in the mouse bone marrow system are mesenchymal. Our results confirm those of Ward and Harris (1976) on the essential role of the 3-hydroxy-4-oxo moiety in the activity of mimosine and related compounds but differ in defining the role of the side chain. Of the compounds tested in our system, only those with an IX-alanine or 2-aminoethyl side chain were active. The synthesis of DNA may not be the function with which the active compounds primarily interfere. Depression of DNA synthesis could be a reflection of any cytotoxic effects of the various compounds which impair the growth rates of the cultures relative to their controls. Many chemical and biochemical investigations to elucidate the mechanism of action of mimosine have been carried out (for reviews see Hylin 1969; Thompson et al. 1969; Hegarty and Paterson 1973). These have shown that mimosine can affect a number of biochemical constituents and biochemical processes within cells, and 120 M. P. Hegarty et al.

the effects may be complex. At least six mechanisms have been postulated by which mimosine may act on animal cells, and it seems likely that different mechanisms may be important in different cells. As far as we know this is the first structure-activity study with bone marrow cells in liquid culture. The system is sensitive and shows good reproducibility provided the conditions are standardized as described here. It could therefore be useful in screening other cytotoxic substances. These in vitro studies have given definitive information on structure-activity relationships but need to be complemented by investigations on whole animals, as individual compounds may show additional biological properties in vivo. For example, mimosine is a powerful depilatory agent in rats and mice but is not goitrogenic, whilst DHP is a potent goitrogen but exerts only a minor effect on the hair follicles (Hegarty et al. 1976). DHP is a dead-end inhibitor of the enzyme catechol-O-methyl­ transferase in vitro (Borchardt 1973) but it remains to be determined whether this is important in vivo.

Acknowledgments The work carried out in the Pathology Department, University of Melbourne, was supported by a grant from the Sir A. E. Roden White Bequest for which we wish to express our thanks to the Trustees. The authors gratefully acknowledge the generous donation of the compounds listed in the text by Dr K. A. Ward, Dr R. L. N. Harris and Dr F. H. Stewart. We thank Mr Theo stoots for expert technical assistance.

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Manuscript received 17 October 1977