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Effects of DAPI, a new diamidine trypanocide, on the ultrastructure of Trypanosoma rhodesiense
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 6, 1978
Effects of DAPI, a new diamidine trypanocide, on the ultrastructure Trypanosoma rhodesiense
of
J. WILLIAMSON AND DIANE J. MCLAREN National Institute for Medical Research, Mill Hill, London NW7 1AA Following the success of berenil, DANN and his colleagues @ANN et al., 1971) synthesized a number of new trvoanocidal diamidines. of which DAPI (102/198) 14’,6-diamidino-2-phenylindole) (Fig. 1) was found to be one of the most active. Apart from antiparasitic activity, its remarkable fluorescence and binding properties have greatly facilitated detection and separation of yeast mitochondrial DNA (WILLIAMSON & FENNELL, 1975) as well as providing an improved stain for Mycoplasma (RUSSELLet al., 1975).
Fig. 1. DAN
(102/198).
Conditions for studying the effects of DAR1 on rhodesiekse- were as described by MACADAM & WILLIAMSON (1972) exceot that trypanosome samples were fixed and processed for electron microscopy according to MCLAREN (1974). The samples were taken six hours after treatment of infected mice with an intraneritoneal curative dose (4.0 mg/kg) of DAPI. Although DAPI was administered as a CDs0 dose, to ensure some degree of equipotent effect with other diamidines tested earlier on T. rhodesiense (pentamidine, hydroxystilbamidine and berenil) (MACADAM& WILLEAMSON.1972). it vroduced more extensive lesions than the other compounds. Lesions such as kinetoplast (k) DNA condensation were common to all diamidine-treated trypanosomes, but nucleolar fragmentation and cytoplasmic cleft formation, which had occurred only variably with the other- diamidines, appeared consistently after DAR1 treatment; all three lesions were frequently visible in the same section (Fig. 2). The kDNA condensation (Fig. 2) was of the Trypanosoma
Fig. 2. Electron micrograph showing the effects of DAR1 on T. rhodesiense: condensation of kinetoplast DNA (KC), nucleolar fragmentation and segregation (NF), cytoplasmic cleft formation (CC) and membrane alteration (MA).
J. WILLIAMSON
discoid type (DELAIN et al., 1971) found with other diamidines, acriflavine and antrycide. Although physical data on DNA-DAPI binding are not yet available in detail, the structure and inter-amidine group spacing would suggest that the mode of binding is likely to be similar to that of other active diamidines,-i.e. by non-intercalative external attachment to the DNA helix (FESTY et al.. 197%. The kinetoplast mitochondrion _was generally dilated, a change produced by other active diamidines, and showed occasional signs of membrane alteration, such as myelin figure formation and, in one instance, apparent disruption. The matrix, especially in the kinetoplast area, was distinctively electron-lucent (Fig. 2); it has been riuggested that such lucent zone; arise by nuclease digestion of drug-modified kDNA (DELAIN et al., 1971). A comoarable but less extensive effect was recorded prevjously with pentamidine (MACADAM & WILLIAMSON,
1972).
The nucleolus was fragmented, with evidence of segregation (Fig. 2). In most cases, the nucleolar fragments were greatly reduced in size and number compared with the example illustrated, and lucent areas developed in the nucleoplasm. Nuclear chromatin appeared to be reduced or absent and both the nuclear membrane and the cisternae of the endoplasmic reticiulum, were dilated. As with pentamidine, such effects on the nucleolus imply interference with RNA synthesis (SIMARD & BERNHARD, 1966; FARBER, 1971). Focal, and in some cases, generalized ribosome depletion were evident, possibly as a result of decreased ribosomal RNA synthesis; occasional ribosomal aggregation may have been due to diamine disolacement (WALLIS. & 1966). Cytoplasmic cleft formation (Fig. 2) appears to result from drug blockade of unsaturated lonachain fatty acid-uptake and promotion of excess deposition of saturated triglyceride in areas adjacent to endoplasmic reticular membrane (WILLIAMSON & MCLAREN, 1974); identical clefts can be produced in normal rat jejunal cells or in neutrophils by an excess supply of saturated long-chain fatty acids (MCKAY et al., 1967; HAWLEY & GORDON, 1976). The capacity to induce these clefts in trypanosomes appears to be restricted to the faster-acting nucleosides, arsenicals and diamidines. A further characteristic effect of DAPI was the local induction of large numbers of microbodies. The maximum number found in any one trypanosome section was much higher than with most other drugs; only puromycin and cordycepin produced comparable numbers. Possibly these ‘glycosomes’ (OPPERDOES & BORST, 1977) which
carry
the vital
glycolytic enzyme complex, may tend to congregate in areas of cell injury and thus reflect a repair or ‘metabolic assistance’ function (MACADAM & WILLIAMSON,
1974).
Acknowledgement We are indebted to Professor samples of DAPI.
Otto Dann
for
References G., Demant,
E. & Volz,
G.
Dann,
O., Bergen,
661
AND D. J. MCLAREN
(1971). Trypanocide Diamidine des 2-Phenylbenzofurans, 2-Phenyl-indens und 2-Phenylindols. Liebigs Annalen der Chemie, 749, 68-89. Delain, E., Brack, C., Riou, G. & Festy, B. (1971). Ultrastructural alterations of Trypanosomu cruzi kinetoolast induced bv the interaction of a trypanocidal drug (hydroxystilbamidine) with the kinetoolast DNA. 7ournal of Uhrastructural * Research, 37, 200-2118. Farber, E. (1971). Response of cells to inhibition of synthesis of DNA, RNA and protein. Biochemical Pharmacology, 20, 1023-1026. Festy, B., Sturm, J. & Daune, M. (1975). Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association. Biochimica et Biophysics Acta, 407, 24-42. Hawley, H. P. & Gordon, G. B. (1976). The effects of long chain free fatty acids on human neutrophil function and structure. Laboratory Investigations, 34, 216-222. Macadam. R. F. & Williamson, T. (1972). Drug effects ‘on the fine structure- of ‘Tryphnosoma rhodesiense : diamidines. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 897-904. Macadam, R. F. & Williamson, J. (1974). Drug effects on the fine structure of Trypanosoma rhodesiense : suramin, tryparsamide and mapharside. Annals of Tropical Medicine and Parasitology, -_ 68, 301-306.McKay, D. G., Kaunitz, H., Csavossy, I. & Tohnson. R. E. (1967). Electron microscope studies of the absorption of lipids. III. Long chain saturated triglycerides. Metabolism, 16, 137-152. McLaren, D. J. (1974). The anterior glands of adult Necator americanus (Nematoda: Strongyloidea). I. Ultrastructural studies. Znternational Journal of Parasitology, 4, 25-37. Opperdoes, F. R. & Borst, I’. (1977). Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Letters, 80, 360-364. Russell, W. C., Williamson, D. H. & Newman, C. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature, 253,461-462. Simard, R. & Bernhard, W. (1966). Le phenomene de la segregation nucleolaire : specificite d’action de certains antimetabolites. Inrernational Journal of Cancer, 1, 463-479.’ Wallis, 0. C. (1966). The effect of pentamidine on ribosomes of the parasitic flagellate Crithidia (Strigomonas) oncopelti. Journal of Protozoology, 13, 234-239. Williamson, D. H. & Fennell, D. J. (1975). The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods in Cell Biology, 12, 335-35 1. Williamson, J. & McLaren, D. J. (1974). Fatty acid metabolism and drug-induced cytoplasmic clefts in trypanosomes. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 263. Accepted for publication
17th May, 1978.
Effects of DAPI, a new diamidine trypanocide, on the ultrastructure Trypanosoma rhodesiense
of
J. WILLIAMSON AND DIANE J. MCLAREN National Institute for Medical Research, Mill Hill, London NW7 1AA Following the success of berenil, DANN and his colleagues @ANN et al., 1971) synthesized a number of new trvoanocidal diamidines. of which DAPI (102/198) 14’,6-diamidino-2-phenylindole) (Fig. 1) was found to be one of the most active. Apart from antiparasitic activity, its remarkable fluorescence and binding properties have greatly facilitated detection and separation of yeast mitochondrial DNA (WILLIAMSON & FENNELL, 1975) as well as providing an improved stain for Mycoplasma (RUSSELLet al., 1975).
Fig. 1. DAN
(102/198).
Conditions for studying the effects of DAR1 on rhodesiekse- were as described by MACADAM & WILLIAMSON (1972) exceot that trypanosome samples were fixed and processed for electron microscopy according to MCLAREN (1974). The samples were taken six hours after treatment of infected mice with an intraneritoneal curative dose (4.0 mg/kg) of DAPI. Although DAPI was administered as a CDs0 dose, to ensure some degree of equipotent effect with other diamidines tested earlier on T. rhodesiense (pentamidine, hydroxystilbamidine and berenil) (MACADAM& WILLEAMSON.1972). it vroduced more extensive lesions than the other compounds. Lesions such as kinetoplast (k) DNA condensation were common to all diamidine-treated trypanosomes, but nucleolar fragmentation and cytoplasmic cleft formation, which had occurred only variably with the other- diamidines, appeared consistently after DAR1 treatment; all three lesions were frequently visible in the same section (Fig. 2). The kDNA condensation (Fig. 2) was of the Trypanosoma
Fig. 2. Electron micrograph showing the effects of DAR1 on T. rhodesiense: condensation of kinetoplast DNA (KC), nucleolar fragmentation and segregation (NF), cytoplasmic cleft formation (CC) and membrane alteration (MA).
J. WILLIAMSON
discoid type (DELAIN et al., 1971) found with other diamidines, acriflavine and antrycide. Although physical data on DNA-DAPI binding are not yet available in detail, the structure and inter-amidine group spacing would suggest that the mode of binding is likely to be similar to that of other active diamidines,-i.e. by non-intercalative external attachment to the DNA helix (FESTY et al.. 197%. The kinetoplast mitochondrion _was generally dilated, a change produced by other active diamidines, and showed occasional signs of membrane alteration, such as myelin figure formation and, in one instance, apparent disruption. The matrix, especially in the kinetoplast area, was distinctively electron-lucent (Fig. 2); it has been riuggested that such lucent zone; arise by nuclease digestion of drug-modified kDNA (DELAIN et al., 1971). A comoarable but less extensive effect was recorded prevjously with pentamidine (MACADAM & WILLIAMSON,
1972).
The nucleolus was fragmented, with evidence of segregation (Fig. 2). In most cases, the nucleolar fragments were greatly reduced in size and number compared with the example illustrated, and lucent areas developed in the nucleoplasm. Nuclear chromatin appeared to be reduced or absent and both the nuclear membrane and the cisternae of the endoplasmic reticiulum, were dilated. As with pentamidine, such effects on the nucleolus imply interference with RNA synthesis (SIMARD & BERNHARD, 1966; FARBER, 1971). Focal, and in some cases, generalized ribosome depletion were evident, possibly as a result of decreased ribosomal RNA synthesis; occasional ribosomal aggregation may have been due to diamine disolacement (WALLIS. & 1966). Cytoplasmic cleft formation (Fig. 2) appears to result from drug blockade of unsaturated lonachain fatty acid-uptake and promotion of excess deposition of saturated triglyceride in areas adjacent to endoplasmic reticular membrane (WILLIAMSON & MCLAREN, 1974); identical clefts can be produced in normal rat jejunal cells or in neutrophils by an excess supply of saturated long-chain fatty acids (MCKAY et al., 1967; HAWLEY & GORDON, 1976). The capacity to induce these clefts in trypanosomes appears to be restricted to the faster-acting nucleosides, arsenicals and diamidines. A further characteristic effect of DAPI was the local induction of large numbers of microbodies. The maximum number found in any one trypanosome section was much higher than with most other drugs; only puromycin and cordycepin produced comparable numbers. Possibly these ‘glycosomes’ (OPPERDOES & BORST, 1977) which
carry
the vital
glycolytic enzyme complex, may tend to congregate in areas of cell injury and thus reflect a repair or ‘metabolic assistance’ function (MACADAM & WILLIAMSON,
1974).
Acknowledgement We are indebted to Professor samples of DAPI.
Otto Dann
for
References G., Demant,
E. & Volz,
G.
Dann,
O., Bergen,
661
AND D. J. MCLAREN
(1971). Trypanocide Diamidine des 2-Phenylbenzofurans, 2-Phenyl-indens und 2-Phenylindols. Liebigs Annalen der Chemie, 749, 68-89. Delain, E., Brack, C., Riou, G. & Festy, B. (1971). Ultrastructural alterations of Trypanosomu cruzi kinetoolast induced bv the interaction of a trypanocidal drug (hydroxystilbamidine) with the kinetoolast DNA. 7ournal of Uhrastructural * Research, 37, 200-2118. Farber, E. (1971). Response of cells to inhibition of synthesis of DNA, RNA and protein. Biochemical Pharmacology, 20, 1023-1026. Festy, B., Sturm, J. & Daune, M. (1975). Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association. Biochimica et Biophysics Acta, 407, 24-42. Hawley, H. P. & Gordon, G. B. (1976). The effects of long chain free fatty acids on human neutrophil function and structure. Laboratory Investigations, 34, 216-222. Macadam. R. F. & Williamson, T. (1972). Drug effects ‘on the fine structure- of ‘Tryphnosoma rhodesiense : diamidines. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 897-904. Macadam, R. F. & Williamson, J. (1974). Drug effects on the fine structure of Trypanosoma rhodesiense : suramin, tryparsamide and mapharside. Annals of Tropical Medicine and Parasitology, -_ 68, 301-306.McKay, D. G., Kaunitz, H., Csavossy, I. & Tohnson. R. E. (1967). Electron microscope studies of the absorption of lipids. III. Long chain saturated triglycerides. Metabolism, 16, 137-152. McLaren, D. J. (1974). The anterior glands of adult Necator americanus (Nematoda: Strongyloidea). I. Ultrastructural studies. Znternational Journal of Parasitology, 4, 25-37. Opperdoes, F. R. & Borst, I’. (1977). Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Letters, 80, 360-364. Russell, W. C., Williamson, D. H. & Newman, C. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature, 253,461-462. Simard, R. & Bernhard, W. (1966). Le phenomene de la segregation nucleolaire : specificite d’action de certains antimetabolites. Inrernational Journal of Cancer, 1, 463-479.’ Wallis, 0. C. (1966). The effect of pentamidine on ribosomes of the parasitic flagellate Crithidia (Strigomonas) oncopelti. Journal of Protozoology, 13, 234-239. Williamson, D. H. & Fennell, D. J. (1975). The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods in Cell Biology, 12, 335-35 1. Williamson, J. & McLaren, D. J. (1974). Fatty acid metabolism and drug-induced cytoplasmic clefts in trypanosomes. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 263. Accepted for publication
17th May, 1978.