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. 1982 Feb 1;155(2):415–431. doi: 10.1084/jem.155.2.415

Destruction of leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers

M Rabinovitch, J-P Dedet, A Ryter, R Robineaux, G Topper, E Brunet
PMCID: PMC2186595  PMID: 7057140

Abstract

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 μM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

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Selected References

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  1. Adams E. The antibacterial action of crystal violet. J Pharm Pharmacol. 1967 Dec;19(12):821–826. doi: 10.1111/j.2042-7158.1967.tb09550.x. [DOI] [PubMed] [Google Scholar]
  2. Alexander J., Vickerman K. Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. J Protozool. 1975 Nov;22(4):502–508. doi: 10.1111/j.1550-7408.1975.tb05219.x. [DOI] [PubMed] [Google Scholar]
  3. Badwey J. A., Karnovsky M. L. Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem. 1980;49:695–726. doi: 10.1146/annurev.bi.49.070180.003403. [DOI] [PubMed] [Google Scholar]
  4. Bashor M. M. Dispersion and disruption of tissues. Methods Enzymol. 1979;58:119–131. doi: 10.1016/s0076-6879(79)58130-0. [DOI] [PubMed] [Google Scholar]
  5. Boveris A., Stoppani A. O., Docampo R., Cruz F. S. Superoxide anion production and trypanocidal action of naphthoquinones on Trypanosoma cruzi. Comp Biochem Physiol C. 1978;61 100(2):327–329. doi: 10.1016/0306-4492(78)90063-1. [DOI] [PubMed] [Google Scholar]
  6. Bray R. S. Leishmania. Annu Rev Microbiol. 1974;28(0):189–217. doi: 10.1146/annurev.mi.28.100174.001201. [DOI] [PubMed] [Google Scholar]
  7. Chance M. L., Peters W., Shchory L. Biochemical taxonomy of Leishmania. I. Observations on DNA. Ann Trop Med Parasitol. 1974 Sep;68(3):307–316. [PubMed] [Google Scholar]
  8. Chang K. P., Dwyer D. M. Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro. Science. 1976 Aug 20;193(4254):678–680. doi: 10.1126/science.948742. [DOI] [PubMed] [Google Scholar]
  9. Chang K. P. Human cutaneous lieshmania in a mouse macrophage line: propagation and isolation of intracellular parasites. Science. 1980 Sep 12;209(4462):1240–1242. doi: 10.1126/science.7403880. [DOI] [PubMed] [Google Scholar]
  10. Docampo R., Cruz F. S., Muniz R. P., Esquivel D. M., de Vasconcellos M. E. Generation of free radicals from phenazine methosulfate in Trypanosoma cruzi epimastigotes. Acta Trop. 1978 Sep;35(3):221–237. [PubMed] [Google Scholar]
  11. Duchateau A., Zeitoun P., Escaig F., Leclerc A., Guillerm J. F. Separation of epoxy-embedded cell cultures from plastic flasks for electron microscopy. Stain Technol. 1980 Jul;55(4):225–228. doi: 10.3109/10520298009067244. [DOI] [PubMed] [Google Scholar]
  12. Hassan H. M., Fridovich I. Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch Biochem Biophys. 1979 Sep;196(2):385–395. doi: 10.1016/0003-9861(79)90289-3. [DOI] [PubMed] [Google Scholar]
  13. Hisada R., Yagi T. 1-Methoxy-5-methylphenazinium methyl sulfate. A photochemically stable electron mediator between NADH and various electron acceptors. J Biochem. 1977 Nov;82(5):1469–1473. doi: 10.1093/oxfordjournals.jbchem.a131836. [DOI] [PubMed] [Google Scholar]
  14. Mattock N. M., Peters W. The experimental chemotherapy of leishmaniasis. I: Techniques for the study of drug action in tissue culture. Ann Trop Med Parasitol. 1975 Sep;69(3):349–357. [PubMed] [Google Scholar]
  15. Murray H. W. Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages. J Exp Med. 1981 May 1;153(5):1302–1315. doi: 10.1084/jem.153.5.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. NUSSENZWEIG V., SONNTAG R., BIANCALANA A., DE FREITAS J. L. P., AMATO NETO V., KLOETZEL J. Acão de corantes trifenil-metânicos sôbre o Trypanosoma cruzi in vitro; emprêgo da violeta de genciana na profilaxia da transmissão da moléstia de Chagas por transfusáo de sangue. Hospital (Rio J) 1953 Dec;44(6):731–744. [PubMed] [Google Scholar]
  17. Nathan C., Nogueira N., Juangbhanich C., Ellis J., Cohn Z. Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of Trypanosoma cruzi. J Exp Med. 1979 May 1;149(5):1056–1068. doi: 10.1084/jem.149.5.1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schnyder J., Baggiolini M. Induction of plasminogen activator secretion in macrophages by electrochemical stimulation of the hexose monophosphate shunt with methylene blue. Proc Natl Acad Sci U S A. 1980 Jan;77(1):414–417. doi: 10.1073/pnas.77.1.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Young R. C., Ozols R. F., Myers C. E. The anthracycline antineoplastic drugs. N Engl J Med. 1981 Jul 16;305(3):139–153. doi: 10.1056/NEJM198107163050305. [DOI] [PubMed] [Google Scholar]

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