Action of 1,10-phenanthroline transition metal chelates on P388 mouse lymphocytic leukaemic cells

Chem.-Biol. Interactions, 16 (1977) 89--99 © Elsevier/North-Holland Scientific Publishers, Ltd.

89

ACTION OF 1,10-PHENANTHROLINE TRANSITION METAL CHELATES ON P388 MOUSE LYMPHOCYTIC LEUKAEMIC CELLS *

A. SHULMAN and GLENDA M. LAYCOCK

Unit of Medical Chemistry and T.R. BRADLEY

Department of Physiology, University of Melbourne, Parkville, 3052 (Australia) (Received August 12th, 1976) (Accepted October l l t h , 1976)

SUMMARY

(1) Fully coordinated 1,10-phenanthroline and 2,2'-bipyridine chelates of Ru(II) are lethal in vitro to cultured and ascites P388 mouse lymphocytic leukaemic cells; 1,10-phenanthroline chelates are generally more p o t e n t than corresponding 2,2'-bipyridine compounds, and mixed-ligand (acetylacetonato) monovalent chelates of both series are more active than the corresponding identical-ligand divalent chelates. Lethal potency is greatest for Ru(II) chelates containing highly alkylated ligands. (2) Within two series of tetramethyl-l,10-phenanthroline chelates, the inert Ru(II) and Ni(II) members are less active against P388 cultured and ascites cells than the corresponding more labile chelates of Cu(II), Cd(II), Zn(II), Fe(II) and Co(II); for the ascites cells, the rank order of lethal p o t e n c y of the chelates correlates reasonably well with their anticipated rank order of kinetic reactivity. (3) Repeated subculture of P388 cells in the presence of a mixed-ligand Ru(II) chelate has produced a cell line that shows a stable 10-fold resistance to the chelate; the resistant cell line is selectively cross-resistant to certain Ru(II) identical and mixed-ligand chelates. (4) The presence of a fluorescent Ru(II) chelate has been demonstrated at the surface and within the cytoplasm and nucleus of P388 ascites cells exposed to it either in vitro or in the mouse. * This work was commenced in the Department of Physiology, University of Melbourne. Present addresses: A.S. and G.M.L.: School of Chemistry, University of Melbourne, Parkville, 3052 (Australia); T.R.B.: Biological Research Unit, Cancer Institute, Melbourne, 3000 (Australia).

90 (5) Ru(II) and Cu(II) chelates of tetramethyl-l,10-phenanthroline do not appear to be chemotherapeutically active against P388 ascites cells in the mouse.

INTRODUCTION Fully coordinated transition metal chelates of 1,10-phenanthroline and related bases have widespread biological [1--8] and clinical [9,10] actions including lethality to Landschiitz ascites tumour cells both in vitro and in the mouse [11,12]. Special attention has been given to ruthenium(II) chelates of 1,10-phenanthroline bases since it has been shown that tris (1,10phenanthroline) l°6Ru(II) perchlorate passes unchanged through the intact animal [13]. Consequently, the biological actions of such highly inert chelates are almost certainly due to the intact chelate cation, and not to its constituent metal ion or ligand [1]. One such antineoplastic Ru(II) chelate, which is also fluorescent, penetrates Landschiitz tumour cells and localizes at the cell surface and in mitochondria and other intracellular structures including the nucleus [12]. Other types of metal complexes active against neoplastic cells have been reported [14,15]. This paper describes the lethal action in vitro of a series of fully coordinated 1,10-phenanthroline and 2,2'-bipyridine chelates of Ru(II)and related transition metal ions against P388 mouse lymphocytic leukaemic cells as well as the action of selected chelates against this tumour in the mouse. The cross-resistance shown by a chelate-resistant subline of P388 to selected phenanthroline chelates differing either in the metal ion or ligand, and the localization of fluorescent Ru(II) phenanthroline chelate in P388 ascites cells are also reported.

MATERIALS AND METHODS

Chelates The following fully coordinated compounds were used in this study: (i) doubly charged Ru(II) chelates containing three identical ligands of 1,10phenanthroline (phen) or 2,2'-bipyridine (bpy) or of 5-methylphen, 5-chlorophen, 5-nitrophen, 4,7
91 anion of all Ru(II) chelates was chloride, of Fe(II) and Ni(II) was sulphate while in all other cases it was acetate. All chelates were completely ionized in solution and sterile, aqueous stock solutions (10 -3 M) were stored at 4°C.

P388 cell line All experiments were carried o u t using either a cultured or an ascites cell line isolated from P388, a mouse l y m p h o c y t i c leukaemia [16]. Optimal conditions for nutrition and growth of dispersed cell suspensions of P388 into discrete clones have been described [17]. P388 cell lines were grown at 37°C in Eagle's basal medium supplemented with 1-serine (0.2 mM), sodium pyruvate (1 mM) and whole calf serum (5%).

Lethal action o f metal chelates to P388 cells Cultured cell line. Inhibition of P388 cell growth by metal chelates was assessed by the m e t h o d of Roosa, Bradley, Law and Herzenberg [18]. Suspensions of approx. 300--500 cells were inoculated into pyrex petri dishes (60 mm diameter) containing doubling dilutions of the test compounds. The dishes were incubated at 37°C in a humid atmosphere of 5% CO2 and 95% air for 7 days and the plating efficiency determined in at least four separate experiments. The lethal titre of the chelate is defined as the lowest concentration which produced zero plating efficiency, i.e., 100% kill of the inoculated cells. Ascites cells. P388 ascites cells, freshly aspirated from the abdominal cavity of a CD/F1 mouse, were centrifuged, washed and suspended in Eagle's supplemented medium together with selected phenanthroline chelates (1 • 10 -s M) for 6 h at 37°C. Samples containing approx l 0 s cells/ml were then diluted with an equal volume of water-soluble nigrosin (1% dissolved in the culture medium) and quadruplicate viable and dead cell counts were carried o u t in a h a e m o c y t o m e t e r chamber; the dead cells were stained black by the penetrating dye. Development o f a P388 chelate-resistant cell line A large population of P388 cultured cells was exposed to the highly active, mixed-ligand chelate acetylacetonato bis(3,4,7,8-tetramethylphen)Ru(II) chloride (RuATMP) (3 • 10 -7 M). The few surviving cells which grew at this concentration were subjected to increasing chelate concentrations until a population was obtained that was maximally resistant at a concentration of 3 • 10 -~ M. This resistant population was then grown in Eagle's supplemented medium in the absence of chelate and the susceptibility of the cells to the chelate tested repeatedly over a period of several months. The lethal titre remained unchanged at 3" 10-6M implying that a stable chelate-resistant P388 cell line had been produced. A small number of stable miniature clones was observed regularly during the development of the resistant P388 cell line. These were n o t investigated further.

92 Cross-resistance o f the chelate-resistant P388 cell line The sensitivity of the RuATMP-resistant P388 cell line to a variety of identical- and mixed-ligand chelates was compared with that of the parental line. Both cell lines were incubated with test chelate for 7 days at 37°C as described previously. Quadruplicate estimations were carried o u t for each concentration of each chelate and all experiments were repeated at least twice. Titres for each c o m p o u n d did n o t vary by more than two-fold.

Chelate localization in P388 ascites cells The fluorescent chelate tris(3,4,7,8-tetramethylphen)Ru(II) chloride (4.2 mg/kg) was administered intraperitoneaUy to C D / F I mice when P388induced ascites was evident. Cells were aspirated some 30--60 min later, washed in physiological saline and examined by fluorescence microscopy for the presence of chelate. In related experiments, freshly aspirated and washed cells were suspended in Eagle's supplemented medium and exposed to the chelate (1 • 10 -4 M) for two h at 37°C in the humidified atmosphere. The cells were then washed in physiological saline and examined by fluorescence microscopy.

Action o f chelates on P388 ascites cells in the mouse Groups of 10 male CD/F1 mice (20--30 g) were inoculated intraperitoneally with approx. 106 freshly aspirated P388 ascites cells. Up to four daily inixaperitoneal injections of selected chelates were c o m m e n c e d 24 h later. The chelates, prepared in physiological saline and administered at the maxim u m dosages tolerated, were tris(3,4,7,8-tetramethylphen)Ru(II) chloride (1.8--2.5 mg/kg), tris(3,5,6,8-tetramethylphen)Ru(II) chloride (2.9--4.2 mg/kg), bis(3,4,7,8-tetramethylphen)Cu(II) acetate (2.8--3.9 mg/kg), and bis(3,5,6,8-tetramethylphen)Cu(II) acetate (2.8--3.9 mg/kg). A control group received saline. The animals were weighed daily and the time of death was recorded.

RESULTS AND DISCUSSION Lethal action o f metal chelates to P388 cells The lethal titres of the series of identical-ligand transition metal chelates of 3,4,7,8-tetramethylphen and 3,5,6,8-tetramethylphen following 7 days contact with cultured P388 cells and the percentages of dead P388 ascites cells produced b y their exposure for 6 h to this series of chelates (1 • 10 -s M) are both shown in Table I. It may be seen for the cultured cells that the Cu(II), Cd(II), Zn(II), Fe(II), and Co(II) chelates of both ligands were approximately equipotent and significantly more active than the corresponding Ni(II) and Ru(II) chelates. The lower p o t e n c y of the Ni(II) and

93 TABLE I LETHAL ACTION OF 1,10-PHENANTHROLINE TRANSITION T O P388 C E L L S - I N - C U L T U R E a A N D A S C I T E S C E L L S b

Test of lethality

METAL

CHELATES

Divalent m e t a l in chelate c Cu

Cd

Zn

Fe

Co

Ni

Ru

3 , 4 , 7 , 8 - T e t r a m e t h y l p h e n d chelates Lethaltitre a Percentage kill in 6 h b

6.0 100

6.1 6.1 6.2 6.1 40 12 15 28

3 , 5 , 6 , 8 - T e t r a m e t h y l p h e n chelates Lethaltitrea Percentage kill in 6 h b

6.2 100

6.3 6.3 6.3 6.2 5.8 5.7 96 89 11 10 17 6

5.5 5.5 6 6

a Cells-in-culture incubated with chelate for 7 days at 37°C in Eagle's s u p p l e m e n t e d m e d i u m containing 5% foetal calf serum. Lethal titres expressed as --logl0M. S.E. = -+0.3. b Aseites cells incubated with chelate (1 • 10 -s M) for 6 h at 3 7 ° C in Eagle's supplem e n t e d m e d i u m t h e n stained with water-soluble nigrosin (1%). Figures indicate percentage of dead cells. c Chelates o f Cu(II), Cd(II), and Zn(II) generally contain t w o identical ligands whereas those of Fe(II), Co(II), Ni(II), and R u ( I I ) contain three. The anion of the Ru(II) chelates is chloride, o f the Fe(II) and Ni(II) chelates is sulphate and of all o t h e r chelates is acetate. d Phen = 1,10-phenanthroline.

Ru(II) chelates is similar to that found for their inhibition of virus multiphcation in chick chorioallantoic membrane [5] and m a y reflect their greater inertness relative to that of the more labile Cu(II), Cd(II), Zn(II), Fe(II), and Co(II) c o m p o u n d s [19]. It may be seen within both series of tetramethylphen chelates shown in Table I that the various members killed P388 ascites cells with different efficacies. As with the cultured cell line, the Ni(II) and Ru(II) c o m p o u n d s were also the least active against the ascites cells. Indeed, with the ascites cells the rank order of lethal p o t e n c y for the tetramethylphen chelates correlated reasonably well with the anticipated rank order of their kinetic reactivities, namely, Cu(II) > Cd(II) > Zn(II) > Fe(II) > Co(II) > Ni(II) > Ru(II) [5, 19]. Similar conclusions have been reached concerning the action of these chelates in a variety of microbial [3--5] and pharmacological systems [7,8] as well as on cells-in-culture [20] and ascites turnout cells,both Landschfitz [11] and mouse plasmacytoma ( M O P C 315). The M O P C 315 ascites cells (10 ~ cells/ml) were incubated in duplicate experiments with each of a series of 3,4,7,8-tetramethylphen chelates at 37°C in Eagle's supplemented medium; the percentage of dead cells was estimated in a haemocytometer chamber after staining with water-soluble nigrosin (1%). The mean percentages of M O P C 315 cells killed following 2 h contact with the Cu(II), Mn(II), Zn(II), Fe(II), Ru(II), Ni(II) and Co(II) chelates (1 • 10 -4 M) were 98, 80, 72, 63, 54, 44, and 20, respectively. It m a y also be seen (Table I) that the Cd(II) and Ni(II) chelates of 3,5,6,

94 TABLE II LETHAL ACTION OF IDENTICAL-LIGAND AND MIXED-LIGAND RU TH EN I U M (II) CHELATES TO P388 CELLS-IN-CULTURE a Predominant ligand

Ru(II) chelate Identical ligand

Phen c 5-Methylphen 5-Chlorophen 5-Nitrophen 4,7-Dimethylphen 3,4,7,8-Tetramethylphen 3,5,6,8-Tetramethylphen 3,4,5,6,7,8-Hexamethylphen Bpy c 4,4'-Dimethylbpy 4,4'-Diethylbpy

<4.3 4.5 5.1 4.3 4.6 5.5 5.7 5.5 <4.3 <4.3 <4.3

Mixed ligand b 5.5 -6.1 5.0 5.3 6.5 6.5 <5.0 5.3 6.3

a Incubated with chelate for 7 days at 37°C in Eagle's supplemented medium containing 5% foetal calf serum. Lethal titres expressed as --togl0M. S.E. -- +0.3. The anion of all chelates is chloride. b Containing one acetylacetonato and two phen or bpy ligands. c Phen-- 1,10-phenanthroline; bpy = 2,2'-bipyridine.

8-tetramethylphen were significantly more active than the corresponding 3,4,7,8-tetramethylphen compounds while the reverse was the case for the Co(II) chelates. The reasons for these differences are not known at present. The lethal titres of related pairs of identical- and mixed-ligand Ru(II) chelates to cultured P388 cells are shown in Table II. It may be seen that the mixed-ligand Ru(II) chelates were much more active than corresponding compounds containing identical ligands. This was also the case for P388 ascites cells following their contact for 6 h with corresponding identical- and acetylacetonato mixed-ligand Ru(II) chelates (1" 10 -s M) of phen, 4,7dimethylphen, 3,4,7,8 -tetramethylphen and 3,5,6,8 -tetramethylphen. Thus, the percentage of dead ascites cells following their exposure to the identicalligand chelates were 1, 0, 6 and 6, respectively, while the corresponding mixed-ligand chelates killed 15, 20, 100 and 100% of P388 cells, respectively. Generally, for both classes of Ru(II) chelates (Table II), lethal potency was greatest in compounds containing tetramethylphen, hexamethylphen, and, to a lesser extent, chlorophen ligands. Moreover, the phen chelates were in the main more active than corresponding, less lipophilic bpy compounds. The greater activity of the mixed-ligand chelates to both P388 cell lines may be due in part to their lower Coulombic charge. However, the two types of chelates no doubt also differ in their oxidation-reduction potentials [21] and in other biologically important properties such as kinetic reactivity; it

95 seems likely that the mixed-ligand chelates would be more labile than corresponding identcal-ligand compounds since the field strength of the acetylacetonato ligand would be less than those of phen or bpy. It is tempting to speculate that the most active chelates in this series are those which combine low peripheral charge and considerable lipophilia and kinetic reactivity which would favour their ready penetration to and interaction with susceptible sites on or within the P388 cells. While precise chemical data are not yet available to justify this suggestion, the much more potent and rapid inhibition of virus multiplication within chick chorioallantoic cells by acetylacetonato bis(3,5,6,8-tetramethylphen)Ru(II) chloride than by the corresponding identical-ligand chelate [22] is consistent with this view.

Cross-resistance o f the chelate-resistant P388 cell line The titres of selected phen chelates required to kill the parent and the derived P388 cell line resistant to RuATMP (3 • 10 -6 M) are shown in Table III. The derived cell line showed equivalent 10-fold resistance to RuATMP and its 3,5,6,8-isomer. However, it was more resistant (40--100 times) to the corresponding tetramethylphen identical-ligand Ru(II) chelates as well as to the mixed-ligand Ru(II) chelates of phen (>33 times) and 5-chlorophen (60 times). On the other hand, notable cross-resistance did not occur to identicalligand Ru(II) chelates of phen, 5-methylphen, 5~hlorophen, 5-nitrophen, 4,7
TABLE III CROSS RESISTANCE OF A CHELATE-RESISTANT P388 CELL LINE a TO IDENTICAL-LIGAND (IL) AND MIXED-LIGAND (ML) RUTHENIUM (II) CHELATES Ligand

Phen e 5-Chlorophen 3,4,7,8-Tetramethylphen 3,5,6,8-Tetramethylphen

Lethal titres b

Degree of resistance

Parent line

Resistant line

IL ¢

IL

<4.3 5.1 5.5 5.7

ML d 5.5 6.1 6.5 6.5

<4.3 5.1 3.5 4.1

ML <4.0 4.3 5.5 5.5

IL ? 0 100 40

cross

ML >33 60 10 10

a Resistant to acetylacetonato bis(3,4,7,8-tetramethylphen)ruthenium(II) chloride 3.10-~M). Both parent and resistant cell lines were incubated with test compound for 7 days at 37°C in Eagle's supplemented medium containing 5% foetal calf serum. Lethal titres expressed as --logl0M. S.E. + 0.3. c Doubly charged chelates containing three identical ligands. d Singly charged chelates containing one acetylacetonato ligand and two identical phen ligands. e Phen = 1,10-phenanthroline.

96 Fe(II), Co(II), and Ni(II) identical-ligand chelates of 3,4,7,8- and 3,5,6,8tetramethylphen. Such resistance and cross-resistance could represent either selection by chelate of a naturally occurring resistant species or chelate-induced modification if the genetic apparatus of the P388 cell. The latter process seems more likely for microorganisms such as Staphylococcus aureus, Saccharomyces cerevisiae, Candida albicans, and Escherichia coli which on rare occasions m a y show as much as a 16-fold resistance to these substances and which produce miniature colonies following exposure to tetramethylphen transition metal chelates (refs. 1,4 and unpublished observations). The develo p m e n t of no more than a 10-fold resistance by P388 cultured cells to RuATMP, the production of miniature P388 clones following exposure to RuATMP and the localization of the active, fluorescent Ru(II) chelate of 3,4,7,8-tetramethylphen in the nucleus of P388 and Landschiitz ascites t u m o u r cells (loc. cit. and ref. 12) are more consistent with the view that development of resistance and of cross-resistance represents an inductive rather than a selective process. Whatever the mechanism of cross-resistance shown by the RuATMP-resistant P388 cell line, it appears to be highly selective for certain Ru(II) chelates. The basis of this selectivity is at present unknown.

Chelate localization in P388 ascites cells Fluorescence microscopic examination of P388 cells aspirated from the abdominal cavity of ascitic mice some 30--60 min after the intraperitoneally administration of tris(3,4,7,8-tetramethylphen)Ru(II) chloride showed evidence of surface chelate adsorption similar to that observed in Landschiitz ascites turnout cells treated in a like manner [12]. The localization of the fluorescent chelate in freshly aspirated P388 cells exposed to it (1 • 10-4 M) in vitro for 2 h is shown in Fig. 1. As with Landschiitz ascites cells [12], the chelate was readily visible both on the surface and within the cytoplasm and nucleus of the P388 cells, the majority of which appeared to be viable at this time (see Table I); similar observations were made with MOPC 315 mouse plasmacytoma cells exposed to the fluorescent chelate under like conditions. Chelate~ontalning structures, apparently radiating w i t h o u t interruption from the nucleus to the cell surface, were also observed in the P388 cells (see cell lower right of Fig. 1) suggesting the presence of a direct structural and presumably functional link between components of the cell nucleus and the cell surface membrane. It is possible that both the surface and nuclear components which bind the fluorescent chelate are DNA. Such a suggestion is consistent with the nature and apparent continuity of the structures concerned and the observation of Rosenberg [15] that t u m o u r cells contain discrete loci of surface membrane DNA both of which bind cis-dichlorodiammineplatinum(II) and related complexes. The ability of the fluorescent chelate to bind initially at discrete sites on the surface and subsequently to mitochondrial and nuclear

97

Fig. 1. F l u o r e s c e n c e in cells f r o m ascites fluid o f m i c e b e a r i n g P 3 8 8 l y m p h o c y t i c leuk a e m i a e x p o s e d f o r 2 h in v i t r o t o t r i s ( 3 , 4 , 7 , 8 - t e t r a m e t h y l - 1 , 1 0 - p h e n a n t h r o l i n e ) - r u t h e n i u m c h l o r i d e (1 • 1 0 -4 M). M o n o c h r o m a t i c i n c i d e n t r a d i a t i o n 4 0 0 - - 4 1 0 m p . E x c i t a t i o n filters 4 m m B G 1 2 ; 4 m m B G 3 8 . Barrier filter 5 3 0 ~ m . M a g n i f i c a t i o n X 250.

c o m p o n e n t s of mouse Landschfitz ascites t u m o u r cells [12] and to induce th e f o r m a t i o n of a wide range of microbial variants in Staph. aureus, E. coli, S. cerevisiae, C. albicans and related microorganisms [1,4] is also consistent with an action on DNA. However, the fluorescent chelate also binds strongly to surface c o m p o n e n t s of guinea pig atrium [7] which are probably muco-

98 p o l y s a c c h a r i d e in n a t u r e a n d , like D N A , c o n t a i n a large n u m b e r o f n e g a t i v e l y c h a r g e d sites w h i c h w o u l d be e x p e c t e d t o b i n d c a t i o n s s t r o n g l y [ 2 3 ] .

A c t i o n o f chelates on P388 ascites cells in the mouse Under the experimental conditions employed, neither the Cu(II) nor the R u ( I I ) c h e l a t e s o f 3,4,7,8- a n d 3 , 5 , 6 , 8 - t e t r a m e t h y l p h e n significantly i n c r e a s e d t h e survival t i m e o f C D / F 1 m i c e i n o c u l a t e d 2 4 h p r i o r l y w i t h P 3 8 8 ascites cells. T h u s , b o t h t h e C u ( I I ) a n d R u ( I I ) chelates, w h i c h w e r e c h e m o t h e r a p e u t i c a l l y active against L a n d s c h t i t z ascites cells in B A L B / C m i c e [ 1 1 ] , w e r e i n e f f e c t i v e u n d e r similar c o n d i t i o n s against P 3 8 8 ascites cells in C D / F 1 m i c e . T h e r e a s o n f o r this d i f f e r e n c e in c h e l a t e a c t i o n is u n k n o w n b u t since b o t h cell lines s h o w e d similar sensitivities t o t h e s e c o m p o u n d s in v i t r o it m a y r e f l e c t d i f f e r e n c e s in t u r n o u t g r o w t h p a t t e r n s in vivo or in t h e d i s p o s i t i o n o f t h e c h e l a t e s in t h e d i f f e r e n t strains o f m i c e . ACKNOWLEDGEMENTS We are m o s t g r a t e f u l t o t h e late P r o f e s s o r F.P. D w y e r a n d M o n s a n t o A u s t r a l i a L i m i t e d f o r t h e m e t a l c h e l a t e s u s e d in this s t u d y a n d t o Mr. J. S m i t h f o r t h e p h o t o g r a p h y . We t h a n k t h e N a t i o n a l H e a l t h a n d Medical R e s e a r c h Council of Australia, the Anticancer Council of Victoria and the Joseph Herman Trust of the University of Melbourne for support. REFERENCES 1 A. Shulman and F.P. Dwyer, Metal chelates in biological systems, in F.P. Dwyer and D.P. MeUor (Eds.), Chelating Agents and Metal Chelates, Academic Press, New York, 1964, p. 383. 2 F.P. Dwyer, I.K. Reid, A. Shulman, G.M. Laycock and S. Dixon, The biological actions of 1,10-phenanthroline and 2,2'-bipyridine hydrochlorides, quarternary salts and metal chelates and related compounds, I. Bacteriostatic action on selected Grampositive, Gram-negative and acid-fast bacteria, Aust. J. Exp. Biol. Med. Sci., 47 (1969) 203. 3 H.M. Butler, A. Hurse, E. Thursky and A. Shulman, Bactericidal action of selected phenanthroline chelates and related compounds, Aust. J. Exp. Biol. Med. Sci., 47 (1969) 541. 4 A. Shnlman, G. Cade, L. Dumble and G.M. Laycock, The lethal action of 1,10phenanthroline transition metal chelates and related compounds on dermatophytes and Candida albicans, Arzneimittel-Forsch., 22 (1972) 154. 5 A. Shulman and D.O. White, Virostatic activity of 1,10-phenanthroline transition metal chelates: a structure--activity analysis, Chem.-Biol. Interact., 6 (1973) 407. 6 A. Shulman, G.M. Laycock, E.J. Ari~ns and A.R.H. Wigmans, The action of selected metal complexes on receptor systems of isolated organs, Part I. The action of selected ruthenium(II) phenanthroline chelates on the cholinergic mechanism of the rat intestine and guinea-pig ileum, Eur. J. Pharmacol., 9 (1970) 347. 7 H. Grossman, A. Shulman and C. Bell, Action of fully co-ordinated, 1,10-phenanthroline transition metal chelates on the guinea-pig isolated atrium, Experientia, 29 (1973) 1522.

99 8 P.G. Farnworth, A. Shulman and A.T. Casey, Actions of 1,10-phenanthroline transition metal chelates and their constituents on the rat isolated diaphragm preparation, Proc. Aust. Physiol. Pharmacol. Soc., 5 (1974) 200. 9 G. Cade, K.H. Shankly, A. Shulman, R.D. Wright, I.O. Stahle, C.B. MacCibbon and E. Lew-Sang, The treatment of dermatological infections with a manganese phenanthroline chelate, Med. J. Aust., 2 (1970) 304. 10 H.M. Butler, J.C. Laver, A. Shulman and R.D. Wright, The use of phenanthroline metal chelates for the control of topical infections due to bacteria, fungi and protozoa, Med. J. Austr., 2 (1970) 309. 11 F.P. Dwyer, E. Mayhew, E.M.F. Roe and A. Shulman, Inhibition of Landschiitz ascites tumour growth by metal chelates derived from 3,4,7,8-tetramet.hyl-l,10-phenanthroline, Brit. J. Cancer, 19 (1965) 195. 12 E. Mayhew, E.M.F. Roe and A. Shulman, Microscopical observations on the effects of phenanthroline chelates on Landschiitz ascites turnout cells, J. Roy. Microsc. Soc., 84 (1965) 475. 13 J.H. Koch, W.P. Rogers, F.P. Dwyer and E.C. Gyarfas, The metabolic fate of tris-l,10phenanthroline l°6ruthenium(II), a compound with anticholinesterase and curare-like activity, Aust. J. Biol. Sci., 10 (1957) 342. 14 K. Takamiya, Anti-tumour activities of copper chelates, Nature, 185 (1960) 190, S. Kirschner, Y.-K. Wei, D. Francis and J.D. Bergman, Anticancer and potential antiviral activity of complex irterganic compounds, J. Med. Chem., 9 (1966) 369; B. Rosenberg, L. VanCamp, J.E. Trosko and V.H. Mansour, Platinum compounds: a new class of p o t e n t antitumour agents, Nature, 222 (1969) 385; G.R. Gale, J.A. Howle and E.M. Walker, A n t i t u m o u r and antimitogenic properties of c/s-dichloro(dipyridine)platinum(II), Cancer Res. 31 (1971) 950; E.M. H o d n e t t and W.J. Dunn, Cobalt derivatives of Schiff bases of aliphatic amines as antitumour agents, J. Med. Chem., 15 (1972) 339. 15 B. Rosenberg, Possible mechanisms for the antitumour activity of platinum coordination complexes, Cancer Chemother. Repts Part 1, 59 (1975) 589. 16 C.J. Dawe and M. Potter, Morphologic and biologic progression of a lymphoid neoplasm of the mouse in vivo and in vitro, Am. J. Pathol., 33 (1957) 603. 17 L.A. Herzenberg and R.A. Roosa, Nutritional requirements for growth of a mouse l y m p h o m a in cell culture, Exp. Cell Res., 21 (1960) 430. 18 R.A. Roosa, T.R. Bradley, L.W. Law and L.A. Herzenberg, Characterization of resistance to amethopterin, 8-azaguanine and several fluorinated pyrimidines in the murine l y m p h o c y t i c neoplasm, P388, J. Cell Comp. Physiol. 60 (1962) 109. 19 F. Basolo and R.G. Pearson, Mechanisms of Inorganic Reactions. A Study of Metal Complexes in Solution, Wiley, New York, 1967. 20 D.O. White, A.W. Harris, I.M. Cheyne, M. Shew, A. Shulman and T.R. Bradley, Actions of metal chelates of substituted 1,10-phenanthrolines on viruses and cells, ILL Actions on cultured cells, Aust. J. Exp. Biol. Med. Sci., 47 (1969) 81. 21 D.A. Buckingham and A.M. Sargeson, Oxidation-reduction potentials as functions of donor atom and ligand, in F.P. Dwyer and D.P. Mellor (Eds.), Chelating Agents and Metal Chelates, Academic Press, New York, 1964, p. 237. 22 D.O. White, A.W. Harris and A Shulman, Actions of metal chelates of substituted 1,10-phenanthrolines on viruses and cells, II. Inhibition of viral multiplication, Aust. J. Exp. Biol. Med. Sci., 41 (1963) 527. 23 G.A. Langer, Heart excitation~ontraction coupling, Ann. Rev. Physiol., 35 (1973) 55.