Print version ISSN 0074-0276
Mem. Inst. Oswaldo Cruz vol. 92 no. 4 Rio de Janeiro July/Aug. 1997
Ultrastructural Alterations Induced by Lithium Chloride in DNA-Containing Organelles of a Bat Trypanosome
Vol. 92(4): 513-516
Adriana G Oliveira, Maurilio J Soares*/+, Artur S Pinto
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 2486, 31270-010 Belo Horizonte, MG, Brasil *Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
Key words: bat trypanosome - ultrastructure - lithium chloride - kinetoplast
It is known that lithium ions induce several alterations in a number of biological systems. This monovalent cation stabilizes cytoskeletal elements, such as actin (R Colombo et al. 1991 Biochem J 274: 421-425) and microtubules (DE Burstein et al. 1985 J Cell Biol 101: 862-870), as well as interferes with DNA replication (EH Anwander et al. 1990 Biopolymers 29: 757-769) and inhibits cell cytokinesis (J Becerra & CL Encina 1987 Experientia 43: 1025-1027). Incubation of the trypanosomatid flagellate Herpetomonas samuelpessoai with lithium chloride resulted in inhibition of both cell proliferation and differentiation (CV Nakamura & AS Pinto 1989 Parasitology 99: 193-197). In this work we analyze the effects of lithium on the ultrastructure of a South American bat trypanosome.
The Trypanosoma cruzi-like strain M29-2 was isolated from a bat (Phyllostomus hastatus) collected in Serrania, State of Minas Gerais, Brazil [AS Pinto et al. 1987 Mem Inst Oswaldo Cruz 82 (Suppl. I): 53], and has been characterized by zymodeme and schizodeme analysis (LFM Teixeira et al. 1993 Parasitol Res 79: 497-500). It has been shown that this trypanosome is non-infective to mice (S Hamanaka & AS Pinto 1993 Rev Soc Bras Med Trop 26: 225-230). Culture epimastigote forms have been maintained by weekly transfers at 28oC in brain-heart-infusion (BHI) medium (Difco, USA), supplemented with 10% fetal calf serum and 0.02% hemoglobin. Some cells were grown in BHI medium containing 50, 100 or 200 mM lithium chloride (Sigma Chem. Co., MO, USA). Cell counts were made, by light microscopy with a Neubauer chamber, at two-days intervals, up to the 12th day. The percent growth inhibition relative to control cultures was calculated at the 6th (middle log phase of growth) and 12th (stationary phase of growth) days post-inoculum. Epimastigotes from lithium-treated cultures were harvested at day 6 post-inoculum by centrifugation at 1,500g / 4oC, washed with phosphate buffered saline (PBS), pH 7.2, and then processed for routine transmission electron microscopy (TEM). To evaluate the effects of lithium chloride on the epimastigote-trypomastigote differentiation (metacyclogenesis), which occurs in old cultures, parasites were grown for 15 days in BHI medium and then 50, 100 or 200 mM LiCl was added to the medium. After six days, cells were collected, smears were stained with Giemsa, and the percent of cell differentiation inhibition relative to the control was calculated by light microscopy.
Cultivation of the parasites at 28oC in culture medium containing lithium chloride resulted in inhibition of both growth (Figs 1, 2) and differentiation (Fig. 3). Analysis of parasites grown in the presence of LiCl by TEM showed that Li+ induced severe alterations in the kinetoplast-DNA (k-DNA) and the nucleus. In affected cells, similar effects were observed with all concentrations (50, 100 and 200 mM) of the drug.
The k-DNA networks of the epimastigote forms lost their typical rod-shaped form (Fig. 4) and appeared as highly electron-dense bodies, presenting several morphological alterations, including incomplete fission (Fig. 5) and fragmentation in variable number of segments (Figs 6, 7). Most nuclei lost their round-shaped profile (Fig. 4) and appeared as irregular (Fig. 5), O-shaped or U-shaped structures. Masses of condensed chromatin were found apposed to the nuclear envelope. Both nuclear and k-DNA alterations were frequently observed in the same cell (Fig. 5).
Despite the alterations in the DNA-containing compartments, the parasites presented no signs of cell degeneration, such as membrane disintegration or cytoplasmic swelling, or lysis. Most cell structures, such as subpellicular microtubules, flagella, Golgi complex and reservosomes appeared not to be affected by the drug, at least at the morphological level. Although most cells presented two basal bodies (Figs 5, 6), usually only a single flagellum could be observed, showing that cell division was blocked in an early stage. Preliminary biochemical analysis of the DNA by gel electrophoresis showed that DNA fragmentation (a hallmark of apoptosis) occurred, resulting in a ladder pattern on the gel (data not shown).
Our data suggest that lithium ions, under the growth conditions and concentrations used, act specifically on DNA or DNA-associated proteins of the parasites. Similar ultrastructural alterations in the k-DNA of epimastigotes of T. cruzi were observed after incubation of the cells with the drug hydroxystilbamidine (E Delain et al. 1971 J Ultrastruct Res 37: 200-217).
It is known that lithium ions bind to nucleic acid bases, leading to an increased H-bond stability (Anwander et al. loc. cit.), which would interfere with DNA replication. Therefore, the alterations observed in both the nucleus and the k-DNA could be explained as aborted attempts for duplication. Alternatively, it has been also described that lithium ions are powerful competitors of mono- and divalent cations, mainly sodium, magnesium and calcium (C Hori & T Oka 1979 Proc Natl Acad Sci USA 76: 2823-2827, K Ptashane et al. 1980 J Cell Physiol 103: 41-46). These cations are regulators of cell proliferation, acting on enzymes involved in initiation of DNA synthesis (H Rubin 1975 Proc Natl Acad Sci USA 72: 3551-3555, E Rosengurt & S Mendonza 1980 Ann N Y Acad Sci 339: 175-190). Therefore, enzymes such as DNA polymerase and DNA topoisomerases I and II could be the target for the lithium activity. It has been shown by in situ hybridization to the Crithidia fasciculata kinetoplast that these enzymes are components of peripheral complexes involved in k-DNA replication (M Ferguson et al. 1992 Cell 70: 621-629), and that T. cruzi proliferation and differentiation are blocked by topoisomerase inhibitors (M Gonzalez-Perdomo et al. 1990 Antimicrob Agents Chemother 34: 1707-1714).
Besides inhibiting either cell growth or differentiation, it has been recently shown that Li+ induces apoptosis in rat immatures cerebellar granule cells (SR D'Mello et al. 1994 Exp Cell Res 211: 332-338). Apoptosis, or programmed cell death (PCD), is a process characterized by DNA fragmentation, detected either at the morphological or biochemical level (EM Johnson & TL Beckwerth 1993 Ann Rev Neurosci 16: 31-46). It has been recently proposed that PCD occurs during the epimastigote-trypomastigote differentiation in T. cruzi, and may participate in the optimal adaptation of the parasite to its different hosts (JC Ameisen et al. 1995 Cell Death Differentiation 2: 285-300). Heat-shock induced apoptosis has been also produced in Leishmania (L.) amazonensis (MEC Moreira et al. 1996 J Cell Physiol 167: 305-313). Considering these data, and the results here presented on the effects of lithium ions on a digenetic trypanosome, together with similar data obtained on an insect trypanosomatid (Nakamura & Pinto loc. cit.), it is possible that in these protozoa these effects involve a direct action of the ion on the DNA assembly, arresting cell division and leading to cell death by apoptosis. Thus, studies on lithium activity might be valuable in elucidating aspects of the regulation of cell proliferation and differentiation among trypanosomatid flagellates, at the cellular and molecular levels.
This work has been supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Pró-Reitoria de Pesquisa (PRPq) da Universidade Federal de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and PADCT/CNPq.
+Corresponding author. Fax: +55-21-260.4434. E-mail: email@example.com
Received 7 October 1996
Accepted 14 March 1997