EFFECTS OF OLIVACINE ON 1B-RS-2 CELL LINESUMMARY
In continuous exposure for 3 days, olivacine hydrobromide was more active than olivacine hydrochloride to inihibit the cell growth of IB-RS-2 pig line. Mitodepressive effect, some ultrastructural changes in mitochondria and nucleoli and some chromosome aberrations were observed in cells exposed to olivacine hydrochloride.
KEY-WORDS
- Olivacine; IB-RS-2 cell line; mitodepressive effect; ultrastructural changes; chromosome aberrations.
RESUMO
Em continua exposigäo durante 3 dias, o hidrobrometo de olivacina foi mais ativo em inibir o crescimento populacional da linhagem celular IB-RS-2 do que o hidrocloreto. Efeito mitodepressivo, algumas alteragöes ultraestructurais em mitocöndrias e nucléolos e algumas aberraqöes cromossömicas foram observadas em células expostas ao hidrocloreto de olivacina.
PALAVRAS-CHAVE
Olivacina; linhagem celular IB-RS-2; efeito mitodepressivo; alteragöes ultraestruturais;aberragöes cromossömicas.
INNTRODUCTION
In the last few years several pyrido (4,3-b) carbazoles compounds, such as the ellipticine and its derivatives endowed with activity "in vivo" against human tumor (7, 8, 11 , ) and currently used in the treatment of metastic breast cancer (9), have been synthetized with the purpose to look for new compounds presenting improved anlicancer properties (5, 10, 17). The action of ellipticine and its derivatives on tumor cells was related to their strong binding to DNA since, the size and the shape of 6H-pyridocarbazole ring, at the intercalation site, allow an almost perfect positioning with a maximum overlap between the heterocyclic ring and the DNA base pairs (6). It was also observed "in vitro" that in some normal or tumor cell systems those compounds induced a decrease of the cell growth rate (14, 15, 23, 24), an inhibition of DNA synthesis (I, 23, 24), an inespecific intracellular alteration (14), some chromosome aberrations (18, 19) and they were highly cytotoxic to cells in specific phases of the cell cycle (1, 2, 22, 23, 24).
Beside ellipticine, its isomer olivacine, (1 ,5-dimethyl6H-pyrido(4,3-b) carbazole), extracted from root barks of Aspidosperma olivaceum, is also endowed with a potent activity "in vitro" against lymphocytic leukemia 1=1210 in murine system (12, 21). However, there are no references about the effects of this compound on normal cell "in vitro".
The present report describes the action of olivacine on cell growth, ultrastructure and chromosomes of an established cell line arised from a normal pig kidney.
MATERIAL AND METHODS
Cell culture: 1B-RS-2 C-26-3 pig cell line was grown at 37.°C in 5% hydrolysate lactoalbumin in Hanks' solution added with 10% inactivated calf serum and pH 7.6 (3, 4).
Olivacine extraction: Root barks of Aspidosperma olivaceum proceeding from the Horto Florestal of Säo Paulo were crumbled and the crude matter was extracted by treatment with ethanol into a soxohlet and concentrated by vacuum evaporation at 50.°C. This crude extract was ressuspended in 10% glacial acetic acid. After resting for 24h at 4.°C it was submitted to a vacuum filtration in order, to remove the residuum. The solute was several times treated with petroleum ether and chloroform, and pH 11 was adjusted with NaOH. After this, it was extracted with chloroform and the chloroform solution was concentrated by vacuum evaporation. The alkaloids were precipitated by adding acetone.
The crude precipitate was purified by silica-gel chromatography. The yield from initial root barks was approximately 0.1%. This alkaloid was transformed in crystaline hydrochloride and hydrobromide.
Effect on cell growth rates: 2 x 105 cells in 5ml of nutrient medium were seeded into prescription bottles and incubated for 24h at 37.°C. After this period, that medium was substituted by a medium containing 0.5, 1.0, 2.0 or 3.0pg/ml of olivacine hydrochloride or hydrobromide and reincubated for 3 days. Every 24h the cells were detached by trypsinization and counted in duplicate. This procedure was repeated 3 times. The cell growth inhibition was estimated by comparison with the growth rates of cells exposed to the olivacines and their respective controls.
Effect on cell ultrastructure: Pellets obtained from cell monolayers, exposed or not to 0.5, I .0, 2.0 and 3.0pg/ml of olivacine hydrochloride for 24, 48 and 72h, were fixed for 12h at 4.°C in 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.0. They were postfixed for 2h at the same temperature in 1% osmium tetroxide in the same buffer, followed by 2h in 2% uranyl acetate. Fixed cells were dehydrated through a graded series of acetone and embedded in Spurr. Thin sections were cut with glass knives in LBK ultramicrotome, placed on carbon-stabilized colodium-coated grids, stained with uranyl acetate and lead citrate and examined in a Philips EM300 electron microscope. (16, 25).
Effect of concentrationn and time of cell-drug contact on mitosis and chromosomes: 1.5 x 105 cells in Iml of nutrient medium were seeded into Leighton tube containing a covership and incubated for 24h at 37.°C. After this period, the medium was poured off and groups of cell monolayers were overlaid for l, 2 and 3h with a medium containig 0.5, 1.0, 2.0 and 3.0pg/ml of olivacine hydrochloride. After these periods of incubation, the cell monolayers were washed once with Hanks' solution to eliminate the drug and each group of incubation was overlaid with medium without drug and reincubated for 1, 2 or 3h. One hour before harvest the cell monolayers, the medium was substituted by medium added Q5 pg/ml of colchicine (NBCo) and incubated at 37.°C. After this period, the cells were submitted for 7 min at 37.°C to hypotonic medium by dilution the medium with prewarmed water ( l :4) and fixated at room temperature for 15 min in Puck's fixative. After hydrolysis for 5min at 60.°C, the cells were stained with 0.2% aqueous fucsin solution and the slides were mounted in Euparal. The inhibition of mitosis induced by olivacine hydrochloride was estimated by counting all mitotic cells present in each cell monolayer compared with their respective controls.
The effect of olivacine hydrochloride on chromosomes was estimated by presence of chromosome aberrations, as clumping, uncoiling, pulverized chromosomes, chromatids breaks, gaps or exchanges, in metaphases. From each cell monolayer, 50 metaphases were at random analysed, but in those samples in which this number was not reached, all present metaphases were examined.
RESULTS
Inhibition of cell growth rates induced by olivacine hydrochloride and hydrobromide: When the growth rates of the cells, which were continuously exposed for 72h to olivacine hydrochloride and hydrobromide, were compared with their respective controls, a decrease of the cell growth rates was related to increase of the dosis employed (Figure l). When the effects of olivacine hydrochloride on the cell growth were compared every 24h with olivacine hydrobromide (Figure 2), the index of cell growth inhibition over 50% at 24h was observed only in the cells exposed to 2 - 3.0pg/ml of olivacine hydrobromide. However, at 48h, this index was also observed in the cells exposed to 2 - 3.0pg/ml of olivacine hydrochloride. Only the cells treated with 0.5pg/ml of olivacine hydrochloride maintained an index under 50% for 72h.
Effects of olivacine hydrochloride (A) and hydrobromide (B) at 0.5, 1.0, 2.0 and 3.0pg/ml on growth of cells exposed for 72h.
Ultrastructure changes induced by olivacine hydrochloride: In the cells exposed to 0.5-3.0pg/ml of olivacine hydrochloride for 3 days, compared with the control were observed ultrastructural changes, which became proportionally accentuated to the concentration of the drug and exposure time. In the cytoplasm of these cells a great number of alterated mitochondria and vacuoles was observed with or without electron dense material. The alterated mitochondria revealed an inner chamber hydropically distended and irregular lucent electron zones instead of a dense matrix. Among them, the most alterated showed a loss of density in the whole matrix space. Usually the inner membrane remainded morphologically intact, except in the last stages of degeneration. The intracrystal space was dilated and sometimes similar to a saccule. Few segments of the crystal membranes had also lost density and usually the distal parts were the most affected. In the nucleous of these cells, the nucleolar body was separated from the condensed surrounding chromatin by a thin rim of less condensed material. Sometimes clear zones were observed in these nucleolus (Figure 3).
Inhibition ofmitosis and chromosome aberrations caused by olivacine hydrochloride When the cell monolayers exposed for 1, 2 and 3h to 0.5, 1.0, 2.0 or 3.0pg/ml of olivacine hydrochloride, followed by a period of nourishing with a medium without this drug, were compared with their respective controls, only those were exposed to 0.5 pg/ml - 2h showed an index of mitosis inhibition under 50%. However, 2 or more hours in a medium without drug were necessary to estimulate mitosis (Figure 4).
Chromosome aberrations were induced in cell monolayers by olivacine hydrochloride at 0.5 - 3.0pg/ml (Figure 5). From the metaphases analysed only the cell monolayers treated with 0.5pg/ml of olivacine hydrochloride revealed the presence of 50% or more of metaphases with normal chromosomes similar to the control (Table 1).
DISCUSSION
The continous exposure of cells, "in vitro", to olivacine hydrochloride and hydrobromide for 3 days revealed a different activity of these salts of olivacine on the cell growth rates„ that was related to the concentrations and the periods of the cell-drug contact. The olivacine hydrobromide was more active in reducing the growth rates of the cells "in vitro" than the hydrochloride form. Similar effect of olivacine hydrochloride was observed, ' 'in vitro" , in mice inoculated with leukemia cells and treated with olivacine hydrochloride annd hydrobromide (OLIVEIRA, M.M. , personal communication, 1986).
The inhibition of cell growth observed "in vitro", mainly With the increase of drug concentrations and the exposure - time, could be due to the cell death induced by some ultrastructural changes in mitochondria and nucleoli and by the presence of a great number of vacuoles, reflecting a derangement of the cell metabolism. Similar ultrastructural alterations as observed in IB-RS-2 cells exposed to olivacine hydrochloride, were also seen in cells exposed to ellipticine (13). However, besides the cell death, the reduction of the cell growth rates induced by olivacine, could also be related to mitodepressive effect, as that observed almost immediately after lh drug-cell contact, on cell monolayers exposed to olivacine hydrochloride at 0.5 - 3.0pg/ml. Similar effect was seen on hamster cells exposed to ellipticine (l). However, the action of olivacine could be mitostatic, similar to that induced by ellipticine (1 ). But this action was dependent of the drug concentration, the period of treatment with the drug and the period of incubation without the drug, as observed in IB-RS-2 cells exposed to 0.5 pg/ml of olivacine hydrochloride for 1 - 2h, followed by nourishment, at least, for 2h with a medium without olivacine. In the other treatments the mitostatic action was not observed.
In all cell monolayers exposed to olivacine hydrochloride were detected metaphases with chromosome aberrations, similar to provoked by ellipticine on hamster cells (26). However, only the group exposed to 0.5 pg/ml of olivacine hydrochloride showed an index over 50% of metaphases with normal chromosomes.
Although olivacine induce some cell damage, the discontinous treatment allows, after ceased the mitostatic action, the growth of normal cells and the elimination of the unviable cells arising from ultrastructural changes and chromosome aberration. Probably, the cell modulation and breaks reversion by repairing, as seen with others pyrido-carbazoles (21), also cooperate with "cures" and increase of the survival time in leukemic animals treated during 30 days with olivacine, every over day (21).
Ultrastructure Of cells exposed to hydrochlonde. A - alterated cell with a great number Of vacuoles (*). B - In the nucleus the nucleolus body is separated from the condensed surrounding chromatin by a thin rim ofless condensed (→). C- Mitochondna show an inner chamber hydropically distended,Focal segments Of the crystal appear with lost density (---->), and sometimes are similar to saccules (→)
Percentage of metaphases with normal and chromosome aberrations in cell monolayers exposed to 0.5, 1.0, 2.0 and 3.0pg/ml of olivacine hydrochloride for l, 2 and 3h and reincubated with a medium without the drug for I . 2 and 3h. Metaphases with one (A) and 2 or more (B) types of hromosome or chromatid abnormalities.
Inhibition of mitosis by olivacine hydrochloride at 0.5 p/ml (A), 1.0pg/ml (B), 2.0pg/ml (C) and 3.0pg/ml (D) in cell monolayers exposed at 37.0 for lh
, 2h 
and 3h 
reincubated with a medium without the drug for 1 , 2 and 3h (exposuretime).
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