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Evaluation of the genotoxic potential of the isocoumarin paepalantine in in vivo and in vitro mammalian systems
Journal of Ethnopharmacology 68 (1999) 115 – 120 www.elsevier.com/locate/jethpharm
Evaluation of the genotoxic potential of the isocoumarin paepalantine in in vivo and in vitro mammalian systems D.C. Tavares a, E.A. Varanda a,*, F.D.P. Andrade b, W. Vilegas b, C.S. Takahashi c a
Department of Biology Science, Faculdade de Cieˆncias Farmaceˆuticas de Araraquara, UNESP, Rodo6ia Araraquara-Jau´ Km 1, 14801 -902, Araraquara, SP, Brazil b Instituto de Quı´mica de Araraquara, UNESP, Rua Professor Francisco Degni, s/n, 14800 -900, Araraquara, SP, Brazil c Department of Biology, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, USP, 14040 -901, Ribeira˜o Preto, SP, Brazil Received 19 February 1999; received in revised form 6 April 1999; accepted 20 April 1999
Abstract Paepalantine is an isocoumarin isolated from Paepalanthus 6ellozioides which showed antimicrobial activity in in vitro experiments. In the present study, paepalantine was tested for possible clastogenic and cytotoxic action. Cultures from different individuals were treated with paepalantine at concentrations of 20, 40 and 80 mg/ml. The effect of isocoumarin was also tested in an in vivo assay using Wistar rat bone marrow cells. Paepalantine was administered intraperitoneally at concentrations of 6.25, 12.5 and 25 mg/kg body weight. Under these conditions paepalantine did not have a clastogenic effect, but was significantly cytotoxic in the in vitro and in vivo mammalian cell systems tested in the present work. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paepalantine; Isocoumarin; Chromosomal aberrations; Cytotoxicity
1. Introduction Reports on plant products and extracts as naturally occurring mutagens are accumulating. For example, cycasin from Cycad nuts has been found to be carcinogenic and mutagenic (Sahu et al., 1981). Furocoumarins such as psoralen derivatives are potent light-activated carcinogens and mutagens (Ames, 1983). Vinblastine and vincristine induce aneuploidy (Luomahaara and Norppa, 1994), while quercetin and several similar * Corresponding author.
flavonoids are mutagens in a number of shortterm systems (Nakayasu et al., 1986; Shimer et al., 1986). A commercial tincture prepared from the herb Ruta gra6eolens L. had a moderate photomutagenic activity when tested at concentrations of up to 2 mg/ml (Schimmer and Ku¨hne, 1990). Isocoumarins are isolated in a great variety of microorganisms, plants, and insects, and have been shown to have considerable biological activity. Some isocoumarins isolated from fungi have an antifungal activity, probably due to competition among the species. The hydroxydihydrocoumarins, produced by Ceratocystis ulmi, cause
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 6 1 - 6
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
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necrotic lesions on pear tree leaves. Some are toxic to man, such as ochratoxin A and ochratoxin B produced by Aspergillus and Penicillum species, while others have beneficial properties: antitumoral (duclauxin), antileukemic (analogue of the bactobolin A), and even antiviral (baciphelacin) activities (Hill, 1986). An isocoumarin, 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c) pyran-1-one, named paepalantine, isolated from Paepalanthus bromelioides (Eriocaulaceae), showed antimicrobial activity in in vitro experiments (Vilegas et al., 1990). Paepalantine was also isolated from P. 6ellozioides and has shown strong mutagenic and cytotoxic activity in the Ames test and Chinese hamster ovary (CHO) cell cultures (Varanda et al., 1997). Paepalantine caused a significant dose-dependent increase in the frequency of revertants in the three strains used in the assay, both with and without S9 mix, in concentrations varying from 2 to 128 mg/ml. The mutagenicity was confirmed in assays with CHO cells treated in G1, S and G2 phases of the cell cycle (Varanda et al., 1997). In view of the potential therapeutic use of paepalantine, the present study was undertaken to evaluate the possible clastogenic effect of this compound in human peripheral blood lymphocytes and bone marrow cells in Wistar rats.
2. Materials and methods
2.1. Chemical agent Paepalantine (Fig. 1) was obtained from P. 6ellozioides following the methodology of Vilegas et al. (1990). The collected plant material was identified as P. 6ellozioides Ruhland (Eriocaulaceae) by Professor Paulo Takeo Sano, Insti-
tuto de Biocieˆncias, Universidade de Sa˜o Paulo. A voucher specimen has been deposited in the herbarium of the Instituto de Biocieˆncias-USP (CFSC13842). The substance was isolated from the chloroformic extract of the capitula of P. 6ellozioides by chromatographic procedures and paepalantine was identified by its spectrometric data (1H NMR, 13C NMR, IR, UV, MS) compared to those already reported in the literature (Vilegas et al., 1990).
2.2. In 6itro test with human peripheral blood lymphocytes Human peripheral blood (lymphocyte-rich plasma) from five healthy non-smoking donors, four females and one male, aged 20–30 years, were grown in RPMI 1640 medium (Sigma) supplemented with 20% fetal calf serum (Cultilab) plus penicillin (Fontoura Wyeth S.A.; 0.005 mg/ ml) and streptomycin (Inlab; 0.01 mg/ml). The cells were stimulated with 2% phytohemagglutinin (Gibco). Lymphocyte cultures were treated with different concentrations of paepalantine 69 h after the beginning of incubation (phase G2 of cell cycle). The paepalantine concentrations tested were 20, 40 and 80 mg/ml. This compound was dissolved in dimethyl sulfoxide (DMSO). The final volume of DMSO used in solvent control cultures was 20 ml/culture. The positive control cultures were treated with doxorubicin (2.5 mg/ml, Farmitalia Carlo Erba, Brazil). The cultures were harvested 72 h after the beginning of incubation. Cochicine (Sigma; 0.04 mg/ml) was added to the cultures 90 min before fixation. Metaphase preparations for analysis of chromosomal aberrations were obtained by the technique of Moorhead et al. (1960). Two thousand cells per culture were counted in each treatment for mitotic index calculation, and 100 metaphases per culture were analyzed in each treatment for chromosomal aberrations.
2.3. In 6i6o test with Wistar rats bone marrow cells
Fig. 1. Paepalantine.
Wistar rats (Rattus nor6egicus) weighing approximately 100 g were treated intraperitoneally
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
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Table 1 Distribution the different types of chromosomal aberrations and mean mitotic index obtained in human lymphocytes from five individuals subjected to different paepalantine treatments at G2 phase of the cell cycle and respective controlsa Treatments (mg/ml)
Control DMSO 20 40 80 DXR (2.5)
Chromosomal aberrations G%
G%%
B%
B%%
Others
11 7 15 26 15 124
1 1 1 4 2 3
4 10 17 12 24 139
2 2 2 4 6 11
0 0 2b 1c, 2d 1b 2e, 2f, 2d
Total
Aberrations/100 cells
AM (%)
MI (%)
18 20 37 49 48 283
3.6 4.0 7.4 9.8 9.6 56.6
3.6 3.6 7.2 9.2 8.4 41.4
5.03 4.69 4.77 3.32 1.87* 5.08
a One hundred cells were analyzed per individual, for a total of 500 cells per treatment. G%, chromatid-type gap; G%%, isochromatid-type gap; B%, chromatid-type break; B%%, isochromatid-type break; AM, abnormal metaphases; MI, mitotic index.; and DXR, positive control with doxorubicin. b Dicentric. c Ring. d Quadriradial figure. e Interchange. f Triradial figure. * Significantly different from the control culture (PB0.05).
with 0.5 ml of the solution 24 h before sacrifice. The rats were divided into six experimental groups of six animals each (three females and three males) as follows: a negative control, a solvent group which received DMSO, a positive control group treated with 30 mg cyclophosphamide/kg body weight (b.w.), and three groups treated with paepalantine at concentrations of 6.25, 12.5 and 25 mg/kg b.w. The animals were injected intraperitoneally with 0.16% colchicine 90 min before sacrifice. The bone marrow preparations for the study of chromosomal aberrations in metaphase cells were obtained by the technique of Ford and Hamerton (1956). One hundred metaphases per animal were analyzed and the mitotic index was calculated for the number of metaphases in 2000 cells analyzed/animal (Preston et al., 1987).
2.4. Statistical analysis The results were analyzed statistically by analysis of variance followed by the Tukey test when it was necessary to determine the levels of the factor between which the difference was significant in the variable studied, with the level of significance set at a= 0.05.
3. Results
3.1. In 6itro test with human peripheral blood lymphocytes Table 1 summarizes the results of chromosomal aberrations analysis in human peripheral lymphocytes in culture following treatment with different concentrations of the isocoumarin paepalantine and in negative, solvent and positive controls. The data obtained showed that the treatments with paepalantine induced an increase in the chromosomal aberrations frequency and in the number of altered cells, which was more noticeable in the 40 and 80 mg/ml concentrations compared with the negative control cultures. However, this increase was not significant in the analysis of variance (P\ 0.05). The most frequently observed chromosomal aberration were chromatid-type gaps and breaks. Dicentrics, rings, triradial and quadriradial figures were the least frequent aberrations. The treatments with paepalantine reduced the mitotic indexes, and with the treatment with the highest paepalentine concentration (80 mg/ml), the mitotic index was significantly reduced compared with the other treatments.
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3.2. In 6i6o test with Wistar rats bone marrow cells The results obtained for the treatments with 6.25, 12.5 and 25 mg paepalantine/kg b.w. and their respective controls are listed in Table 2. Animals treated with different concentrations of paepalantine did not show significant differences in chromosomal aberration frequency or in the number of abnormal metaphases compared to animals not submitted to treatment (negative control). The analysis of chromosomal aberrations obtained in the different treatments showed that the most frequent types of aberration were chromatid-type gaps and breaks. Lower values for the mitotic index were observed in the animals treated with the different paepalantine doses compared with the negative control animals. The reduction was statistically significant in the treatments with 12.5 and 25 mg/kg b.w. paepalantine compared with the mitotic indexes observed in the control group.
4. Discussion The composition of the standard battery of the tests is necessary to define the genotoxic potential of proposed new drugs. Both Europe and Japan require tests for gene mutations in bacteria, chro-
mosomal aberrations in mammalian cells, and in vivo mammalian cell tests to detect chromosomal damage (Tice et al., 1994; Purves et al., 1995). The present study also assessed the genotoxic activity of paepalantine in human lymphocytes from peripheral blood in culture and in Wistar rat bone marrow cells. As it was carried out on mammals, the data obtained from clastogenicity tests in vitro should be a priori easier to extrapolate to man. Therefore, human cells should be preferred to animal cells for the following reasons: they have been more widely investigated not only for established mutagens–carcinogens but also for a lot of pharmaceuticals; there is a closer karyotypical analogy from which it can, in general, expect a similar sensitivity or resistance to the drugs tested; though differences can occur, the similarity of metabolism is certainly greater in the cells themselves, excluding added microsomal fractions which are generally from an animal origin (Moutschen, 1980). In the present work, the in vitro experiments for the evaluation of chromosomal aberration frequency showed that paepalantine had no statistically significant clastogenic effect. Since chromosomal aberrations arise as a result of misrepaired or unrepaired lesions, the inhibition of G2 repair is shown to give rise to an increase in chromosomal aberrations, which correspond to the lesions
Table 2 Distribution the different types of chromosomal aberrations and mean mitotic index obtained in rats bone marrow cells from six animals subjected to different paepalantine treatments and respective controlsa Treatments (mg/kg b.w.)
Control DMSO 6.25 12.50 25.00 CP (15.00) a
Chromosomal aberrations G%
G%%
B%
B%%
Others
11 4 7 12 7 25
0 0 0 0 1 1
3 8 2 6 4 130
0 1 1 1 0 9
0 0 0 0 0 6b
Total
Aberrations/100 cells
AM (%)
MI (%)
14 13 10 19 12 171
2.34 2.17 1.67 3.17 2.00 28.50
2.34 2.17 1.67 3.17 2.00 13.12
5.66 4.06 3.98 2.87* 2.59* 1.10
One hundred cells were analyzed per animal, for a total of 600 cells per treatment. G%, chromatid-type gap; G%%, isochromatidtype gap; B%, chromatid-type break; B%%, isochromatid-type break; AM, abnormal metaphases; MI, mitotic index.; and CP, positive control with cyclophosphamide. b Triradial figure. * Significantly different from the negative control animals (PB0.05).
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
that are normally repaired during the G2 phase (Pincheira et al., 1993). The mutagenic activity of paepalantine in CHO cells was more active in the G2 phase, suggesting a lesion-repair action (Varanda et al., 1997). Thus the genotoxic assessment of paepalantine in human lymphocytes in culture was carried out in the G2 phase of the cell cycle, although no significant clastogenic effect has been found. Results of in vitro tests for induced chromosome aberrations can differ. Many factors may contribute to this variability, including differences in protocols, and possible differences in sensitivities among cell types (Galloway et al., 1997). On the other hand, at the highest test concentration (80 mg/ml), there was a significant reduction in the mitotic index compared with the other treatments. These results suggest that paepalantine is toxic at high concentrations, since at doses of 100 mg/ml or higher, cell growth was inhibited. The cytotoxicity of paepalantine was also shown in CHO and McCoy cells (Varanda et al., 1997). The toxicity could be expected because the carbon skeleton of paepalantine is compatible with the coumarin compounds and the double connection 3–4 seems to be a critical reactive site. However, this compound was 10 times more toxic than coumarin, which according to Fernybough et al. (1994), has an IC50 close to 1 mM, determined by the MTT technique in mouse hepatocytes. According to Galloway et al. (1997) a suitable upper toxicity limit was a reduction in cell growth of \50%. Positive results could be obtained without exceeding a reduction of 50 – 60% in cell growth. Varanda et al. (1997) observed paepalantine clastogenic activity in CHO cells, but there was a simultaneous reduction from 50 to 70% in the mitotic index values. Interpretation of data produced at high levels of mitotic inhibition is not clear. This topic and the need for further data to clarify the relevance of ‘high toxicity clastogens’ has been discussed thoroughly by Kirkland (1992). It is possible, thought not proven, that an increase in aberrations at highly toxic concentrations arises as a result of the toxicity itself. Furthermore, chemicals which induce clastogenicity only at doses which induce significant mitotic inhibition or de-
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lay may be of less biological importance than ‘low toxicity clastogens’ since they are probably highly cytotoxic at these concentrations. The relevance of the clastogenicity of substances which is only seen at very toxic doses is still not clear and awaits further correlation of in vitro and in vivo clastogenicity responses (Henderson et al., 1996). According to Purves et al. (1995), an positive in vivo result defines a genotoxic compound whilst in vitro positives only suggest genotoxic potential in mammalian cells. On this basis, we used the Wistar rats bone marrow test system. Similar to the result obtained in human lymphocytes in culture, the data obtained for the in vivo assay showed no clastogenic effect in the different treatments with paepalantine. Nevertheless, this isocoumarin was cytotoxic, and significantly lower mitotic index values were observed in the treatments with 12.5 and 25 mg/kg b.w. paepalantine when compared with animals not submitted to any treatment. Thus, paepalantine isolated from the P. 6ellozioides plant, did not have a clastogenic effect, but cytotoxicity from this isocoumarin was observed both in vitro and in vivo mammalian systems tested in the study. Alterations in its chemical structure are being made to reduce its adverse effects because of its importance as an antimicrobial agent.
Acknowledgements This research was supported by FAPESP, CNPq, and PADC-FCF-UNESP.
References Ames, N.A., 1983. Dietary carcinogens and anticarcinogens. Science 221, 1256 – 1264. Fernybough, L., Kell, S.W., Hammond, A.L., Thomas, N.W., Fry, J.R., 1994. Comparison of in vivo and in vitro rat hepatic toxicity of coumarin and methyl analogues, and application of quantitative morphometry to toxicity in vivo. Toxicology 88, 113 – 125. Ford, C.E., Hamerton, J.L., 1956. A colchicine hypotonic citrate squash sequence for mammalian chromosomes. Stain Technol. 31, 247 – 251.
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D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120 Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., Shelby, M., 1987. Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells. Mutat. Res. 189, 157 – 165. Purves, D., Harvey, C., Tweats, D., Lumley, C.E., 1995. Genotoxicity testing: current practices and strategies used by the pharmaceutical industry. Mutagenesis 10, 297 – 312. Sahu, R.K., Basu, R., Sharma, A., 1981. Genetic toxicological testing of some plant flavonoids by the micronucleus test. Mutat. Res. 89, 69 – 74. Shimer, O., Ha¨fele, F., Kru¨ger, A., 1986. The mutagenic potencies of plant extracts containing quercetin in Salmonella typhimurium TA98 and TA100. Mutat. Res. 206, 201 – 208. Schimmer, O., Ku¨hne, I., 1990. Mutagenic compounds in an extracted from Rutae Herba (Ruta gra6eolens). II. UV-A mediated mutagenicity in the freen alga Chalmydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba. Mutat. Res. 243, 57 – 62. Tice, R.R., Hayashi, M., MacGregor, J.T., et al., 1994. Report from the working group on the in vivo mammalian bone marrow chromosomal aberration test. Mutat. Res. 312, 305 – 312. Varanda, E.A., Raddi, M.S.G., Dias, F.L., et al., 1997. Mutagenic and cytotoxic activity of an isocoumarin (Paepalantine) isolated from Paepalanthus 6ellozioides. Teratog. Carcionog. Mutagen. 17, 85 – 95. Vilegas, W., Roque, N.F., Salatino, A., Giesbrecht, A.M., Davino, S., 1990. Isocoumarin from Paepalanthus bromelioides. Phytochemistry 29, 2299 – 2301.
Galloway, S.M., Sofuni, T., Shelby, M.D., et al., 1997. Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols. Environ. Mol. Mutagen. 29, 189–207. Henderson, L., Jones, E., Freemantle, M., et al., 1996. Extended harvest times are not necessary for the detection of in vitro clastogens in regulatory cytogenetics studies. Mutagenesis 11, 61 – 67. Hill, R.A., 1986. Naturally occurring isocoumarins. Fortschr. Chem. Org. Naturstoffe 49, 1–78. Kirkland, D.J., 1992. Chromosomal aberration tests in vitro: problems with protocol design and interpretation of results. Mutagenesis 7, 95–106. Luomahaara, S., Norppa, H., 1994. Induction of micronuclei in cultured human lymphocytes treated with vinblastine before and after mitogen stimulation. Mutat. Res. 324, 29– 34. Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M., Hungerford, D.A., 1960. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613 – 616. Moutschen, J., 1980. Cytogenetical test on mammalian cells in vitro. Arch. Toxicol. 46, 61–75. Nakayasu, M., Sakamoto, H., Terada, M., Nagao, M., Sugimura, T., 1986. Mutagenicity of quercetin in Chinese hamster lung cells in culture. Mutat. Res. 174, 79–83. Pincheira, J., Gallo, C., Bravo, M., Navarrete, M.H., LopezSaez, J.P., 1993. G2 repair and aging: influence of donor age on chromosomal aberrations in human lymphocytes. Mutat. Res. 295, 55 –62.
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Evaluation of the genotoxic potential of the isocoumarin paepalantine in in vivo and in vitro mammalian systems D.C. Tavares a, E.A. Varanda a,*, F.D.P. Andrade b, W. Vilegas b, C.S. Takahashi c a
Department of Biology Science, Faculdade de Cieˆncias Farmaceˆuticas de Araraquara, UNESP, Rodo6ia Araraquara-Jau´ Km 1, 14801 -902, Araraquara, SP, Brazil b Instituto de Quı´mica de Araraquara, UNESP, Rua Professor Francisco Degni, s/n, 14800 -900, Araraquara, SP, Brazil c Department of Biology, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, USP, 14040 -901, Ribeira˜o Preto, SP, Brazil Received 19 February 1999; received in revised form 6 April 1999; accepted 20 April 1999
Abstract Paepalantine is an isocoumarin isolated from Paepalanthus 6ellozioides which showed antimicrobial activity in in vitro experiments. In the present study, paepalantine was tested for possible clastogenic and cytotoxic action. Cultures from different individuals were treated with paepalantine at concentrations of 20, 40 and 80 mg/ml. The effect of isocoumarin was also tested in an in vivo assay using Wistar rat bone marrow cells. Paepalantine was administered intraperitoneally at concentrations of 6.25, 12.5 and 25 mg/kg body weight. Under these conditions paepalantine did not have a clastogenic effect, but was significantly cytotoxic in the in vitro and in vivo mammalian cell systems tested in the present work. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paepalantine; Isocoumarin; Chromosomal aberrations; Cytotoxicity
1. Introduction Reports on plant products and extracts as naturally occurring mutagens are accumulating. For example, cycasin from Cycad nuts has been found to be carcinogenic and mutagenic (Sahu et al., 1981). Furocoumarins such as psoralen derivatives are potent light-activated carcinogens and mutagens (Ames, 1983). Vinblastine and vincristine induce aneuploidy (Luomahaara and Norppa, 1994), while quercetin and several similar * Corresponding author.
flavonoids are mutagens in a number of shortterm systems (Nakayasu et al., 1986; Shimer et al., 1986). A commercial tincture prepared from the herb Ruta gra6eolens L. had a moderate photomutagenic activity when tested at concentrations of up to 2 mg/ml (Schimmer and Ku¨hne, 1990). Isocoumarins are isolated in a great variety of microorganisms, plants, and insects, and have been shown to have considerable biological activity. Some isocoumarins isolated from fungi have an antifungal activity, probably due to competition among the species. The hydroxydihydrocoumarins, produced by Ceratocystis ulmi, cause
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 6 1 - 6
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
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necrotic lesions on pear tree leaves. Some are toxic to man, such as ochratoxin A and ochratoxin B produced by Aspergillus and Penicillum species, while others have beneficial properties: antitumoral (duclauxin), antileukemic (analogue of the bactobolin A), and even antiviral (baciphelacin) activities (Hill, 1986). An isocoumarin, 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c) pyran-1-one, named paepalantine, isolated from Paepalanthus bromelioides (Eriocaulaceae), showed antimicrobial activity in in vitro experiments (Vilegas et al., 1990). Paepalantine was also isolated from P. 6ellozioides and has shown strong mutagenic and cytotoxic activity in the Ames test and Chinese hamster ovary (CHO) cell cultures (Varanda et al., 1997). Paepalantine caused a significant dose-dependent increase in the frequency of revertants in the three strains used in the assay, both with and without S9 mix, in concentrations varying from 2 to 128 mg/ml. The mutagenicity was confirmed in assays with CHO cells treated in G1, S and G2 phases of the cell cycle (Varanda et al., 1997). In view of the potential therapeutic use of paepalantine, the present study was undertaken to evaluate the possible clastogenic effect of this compound in human peripheral blood lymphocytes and bone marrow cells in Wistar rats.
2. Materials and methods
2.1. Chemical agent Paepalantine (Fig. 1) was obtained from P. 6ellozioides following the methodology of Vilegas et al. (1990). The collected plant material was identified as P. 6ellozioides Ruhland (Eriocaulaceae) by Professor Paulo Takeo Sano, Insti-
tuto de Biocieˆncias, Universidade de Sa˜o Paulo. A voucher specimen has been deposited in the herbarium of the Instituto de Biocieˆncias-USP (CFSC13842). The substance was isolated from the chloroformic extract of the capitula of P. 6ellozioides by chromatographic procedures and paepalantine was identified by its spectrometric data (1H NMR, 13C NMR, IR, UV, MS) compared to those already reported in the literature (Vilegas et al., 1990).
2.2. In 6itro test with human peripheral blood lymphocytes Human peripheral blood (lymphocyte-rich plasma) from five healthy non-smoking donors, four females and one male, aged 20–30 years, were grown in RPMI 1640 medium (Sigma) supplemented with 20% fetal calf serum (Cultilab) plus penicillin (Fontoura Wyeth S.A.; 0.005 mg/ ml) and streptomycin (Inlab; 0.01 mg/ml). The cells were stimulated with 2% phytohemagglutinin (Gibco). Lymphocyte cultures were treated with different concentrations of paepalantine 69 h after the beginning of incubation (phase G2 of cell cycle). The paepalantine concentrations tested were 20, 40 and 80 mg/ml. This compound was dissolved in dimethyl sulfoxide (DMSO). The final volume of DMSO used in solvent control cultures was 20 ml/culture. The positive control cultures were treated with doxorubicin (2.5 mg/ml, Farmitalia Carlo Erba, Brazil). The cultures were harvested 72 h after the beginning of incubation. Cochicine (Sigma; 0.04 mg/ml) was added to the cultures 90 min before fixation. Metaphase preparations for analysis of chromosomal aberrations were obtained by the technique of Moorhead et al. (1960). Two thousand cells per culture were counted in each treatment for mitotic index calculation, and 100 metaphases per culture were analyzed in each treatment for chromosomal aberrations.
2.3. In 6i6o test with Wistar rats bone marrow cells
Fig. 1. Paepalantine.
Wistar rats (Rattus nor6egicus) weighing approximately 100 g were treated intraperitoneally
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
117
Table 1 Distribution the different types of chromosomal aberrations and mean mitotic index obtained in human lymphocytes from five individuals subjected to different paepalantine treatments at G2 phase of the cell cycle and respective controlsa Treatments (mg/ml)
Control DMSO 20 40 80 DXR (2.5)
Chromosomal aberrations G%
G%%
B%
B%%
Others
11 7 15 26 15 124
1 1 1 4 2 3
4 10 17 12 24 139
2 2 2 4 6 11
0 0 2b 1c, 2d 1b 2e, 2f, 2d
Total
Aberrations/100 cells
AM (%)
MI (%)
18 20 37 49 48 283
3.6 4.0 7.4 9.8 9.6 56.6
3.6 3.6 7.2 9.2 8.4 41.4
5.03 4.69 4.77 3.32 1.87* 5.08
a One hundred cells were analyzed per individual, for a total of 500 cells per treatment. G%, chromatid-type gap; G%%, isochromatid-type gap; B%, chromatid-type break; B%%, isochromatid-type break; AM, abnormal metaphases; MI, mitotic index.; and DXR, positive control with doxorubicin. b Dicentric. c Ring. d Quadriradial figure. e Interchange. f Triradial figure. * Significantly different from the control culture (PB0.05).
with 0.5 ml of the solution 24 h before sacrifice. The rats were divided into six experimental groups of six animals each (three females and three males) as follows: a negative control, a solvent group which received DMSO, a positive control group treated with 30 mg cyclophosphamide/kg body weight (b.w.), and three groups treated with paepalantine at concentrations of 6.25, 12.5 and 25 mg/kg b.w. The animals were injected intraperitoneally with 0.16% colchicine 90 min before sacrifice. The bone marrow preparations for the study of chromosomal aberrations in metaphase cells were obtained by the technique of Ford and Hamerton (1956). One hundred metaphases per animal were analyzed and the mitotic index was calculated for the number of metaphases in 2000 cells analyzed/animal (Preston et al., 1987).
2.4. Statistical analysis The results were analyzed statistically by analysis of variance followed by the Tukey test when it was necessary to determine the levels of the factor between which the difference was significant in the variable studied, with the level of significance set at a= 0.05.
3. Results
3.1. In 6itro test with human peripheral blood lymphocytes Table 1 summarizes the results of chromosomal aberrations analysis in human peripheral lymphocytes in culture following treatment with different concentrations of the isocoumarin paepalantine and in negative, solvent and positive controls. The data obtained showed that the treatments with paepalantine induced an increase in the chromosomal aberrations frequency and in the number of altered cells, which was more noticeable in the 40 and 80 mg/ml concentrations compared with the negative control cultures. However, this increase was not significant in the analysis of variance (P\ 0.05). The most frequently observed chromosomal aberration were chromatid-type gaps and breaks. Dicentrics, rings, triradial and quadriradial figures were the least frequent aberrations. The treatments with paepalantine reduced the mitotic indexes, and with the treatment with the highest paepalentine concentration (80 mg/ml), the mitotic index was significantly reduced compared with the other treatments.
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D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
3.2. In 6i6o test with Wistar rats bone marrow cells The results obtained for the treatments with 6.25, 12.5 and 25 mg paepalantine/kg b.w. and their respective controls are listed in Table 2. Animals treated with different concentrations of paepalantine did not show significant differences in chromosomal aberration frequency or in the number of abnormal metaphases compared to animals not submitted to treatment (negative control). The analysis of chromosomal aberrations obtained in the different treatments showed that the most frequent types of aberration were chromatid-type gaps and breaks. Lower values for the mitotic index were observed in the animals treated with the different paepalantine doses compared with the negative control animals. The reduction was statistically significant in the treatments with 12.5 and 25 mg/kg b.w. paepalantine compared with the mitotic indexes observed in the control group.
4. Discussion The composition of the standard battery of the tests is necessary to define the genotoxic potential of proposed new drugs. Both Europe and Japan require tests for gene mutations in bacteria, chro-
mosomal aberrations in mammalian cells, and in vivo mammalian cell tests to detect chromosomal damage (Tice et al., 1994; Purves et al., 1995). The present study also assessed the genotoxic activity of paepalantine in human lymphocytes from peripheral blood in culture and in Wistar rat bone marrow cells. As it was carried out on mammals, the data obtained from clastogenicity tests in vitro should be a priori easier to extrapolate to man. Therefore, human cells should be preferred to animal cells for the following reasons: they have been more widely investigated not only for established mutagens–carcinogens but also for a lot of pharmaceuticals; there is a closer karyotypical analogy from which it can, in general, expect a similar sensitivity or resistance to the drugs tested; though differences can occur, the similarity of metabolism is certainly greater in the cells themselves, excluding added microsomal fractions which are generally from an animal origin (Moutschen, 1980). In the present work, the in vitro experiments for the evaluation of chromosomal aberration frequency showed that paepalantine had no statistically significant clastogenic effect. Since chromosomal aberrations arise as a result of misrepaired or unrepaired lesions, the inhibition of G2 repair is shown to give rise to an increase in chromosomal aberrations, which correspond to the lesions
Table 2 Distribution the different types of chromosomal aberrations and mean mitotic index obtained in rats bone marrow cells from six animals subjected to different paepalantine treatments and respective controlsa Treatments (mg/kg b.w.)
Control DMSO 6.25 12.50 25.00 CP (15.00) a
Chromosomal aberrations G%
G%%
B%
B%%
Others
11 4 7 12 7 25
0 0 0 0 1 1
3 8 2 6 4 130
0 1 1 1 0 9
0 0 0 0 0 6b
Total
Aberrations/100 cells
AM (%)
MI (%)
14 13 10 19 12 171
2.34 2.17 1.67 3.17 2.00 28.50
2.34 2.17 1.67 3.17 2.00 13.12
5.66 4.06 3.98 2.87* 2.59* 1.10
One hundred cells were analyzed per animal, for a total of 600 cells per treatment. G%, chromatid-type gap; G%%, isochromatidtype gap; B%, chromatid-type break; B%%, isochromatid-type break; AM, abnormal metaphases; MI, mitotic index.; and CP, positive control with cyclophosphamide. b Triradial figure. * Significantly different from the negative control animals (PB0.05).
D.C. Ta6ares et al. / Journal of Ethnopharmacology 68 (1999) 115–120
that are normally repaired during the G2 phase (Pincheira et al., 1993). The mutagenic activity of paepalantine in CHO cells was more active in the G2 phase, suggesting a lesion-repair action (Varanda et al., 1997). Thus the genotoxic assessment of paepalantine in human lymphocytes in culture was carried out in the G2 phase of the cell cycle, although no significant clastogenic effect has been found. Results of in vitro tests for induced chromosome aberrations can differ. Many factors may contribute to this variability, including differences in protocols, and possible differences in sensitivities among cell types (Galloway et al., 1997). On the other hand, at the highest test concentration (80 mg/ml), there was a significant reduction in the mitotic index compared with the other treatments. These results suggest that paepalantine is toxic at high concentrations, since at doses of 100 mg/ml or higher, cell growth was inhibited. The cytotoxicity of paepalantine was also shown in CHO and McCoy cells (Varanda et al., 1997). The toxicity could be expected because the carbon skeleton of paepalantine is compatible with the coumarin compounds and the double connection 3–4 seems to be a critical reactive site. However, this compound was 10 times more toxic than coumarin, which according to Fernybough et al. (1994), has an IC50 close to 1 mM, determined by the MTT technique in mouse hepatocytes. According to Galloway et al. (1997) a suitable upper toxicity limit was a reduction in cell growth of \50%. Positive results could be obtained without exceeding a reduction of 50 – 60% in cell growth. Varanda et al. (1997) observed paepalantine clastogenic activity in CHO cells, but there was a simultaneous reduction from 50 to 70% in the mitotic index values. Interpretation of data produced at high levels of mitotic inhibition is not clear. This topic and the need for further data to clarify the relevance of ‘high toxicity clastogens’ has been discussed thoroughly by Kirkland (1992). It is possible, thought not proven, that an increase in aberrations at highly toxic concentrations arises as a result of the toxicity itself. Furthermore, chemicals which induce clastogenicity only at doses which induce significant mitotic inhibition or de-
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lay may be of less biological importance than ‘low toxicity clastogens’ since they are probably highly cytotoxic at these concentrations. The relevance of the clastogenicity of substances which is only seen at very toxic doses is still not clear and awaits further correlation of in vitro and in vivo clastogenicity responses (Henderson et al., 1996). According to Purves et al. (1995), an positive in vivo result defines a genotoxic compound whilst in vitro positives only suggest genotoxic potential in mammalian cells. On this basis, we used the Wistar rats bone marrow test system. Similar to the result obtained in human lymphocytes in culture, the data obtained for the in vivo assay showed no clastogenic effect in the different treatments with paepalantine. Nevertheless, this isocoumarin was cytotoxic, and significantly lower mitotic index values were observed in the treatments with 12.5 and 25 mg/kg b.w. paepalantine when compared with animals not submitted to any treatment. Thus, paepalantine isolated from the P. 6ellozioides plant, did not have a clastogenic effect, but cytotoxicity from this isocoumarin was observed both in vitro and in vivo mammalian systems tested in the study. Alterations in its chemical structure are being made to reduce its adverse effects because of its importance as an antimicrobial agent.
Acknowledgements This research was supported by FAPESP, CNPq, and PADC-FCF-UNESP.
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