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microsome assay and chromosomal aberrations in CHO cells
Journal of Ethnopharmacology 81 (2002) 257 /264 www.elsevier.com/locate/jethpharm
Genotoxicity of Brosimum gaudichaudii measured by the Salmonella/ microsome assay and chromosomal aberrations in CHO cells Eliana Aparecida Varanda a,*, Gilberto Luiz Pozetti b, Miriam Verginia Lourenc¸o b, Wagner Vilegas b, Maria Stella Gonc¸alves Raddi a a
Departamento de Cieˆncias Biolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas de Araraquara, Universidade Estadual Paulista, UNESP, Rodovia Araraquara Jau´-Km 1, 14801-902 Araraquara, SP, Brazil b Instituto de Quı´mica de Araraquara, Universidade Estadual Paulista, UNESP, Sa˜o Paulo, Brasil. Rua Prof. Francisco Degni s/n, 14801-970 Araraquara, SP, Brazil Received 31 May 2001; received in revised form 31 March 2002; accepted 4 April 2002
Abstract The root bark of Brosimum gaudichaudii Tre´cul (Moraceae) is popularly used for treatment of vitiligo. In the present study the mutagenic activity of the aqueous and methanolic extract as well as of the n -butanolic fraction of this medicinal plant were evaluated using Salmonella typhimurium assays, TA100, TA98, TA102 and TA97a strains, while the clastogenic effect in Chinese hamster ovary (CHO) cells in the G1/S, S and G2/S phases of the cell cycle. The results showed mutagenic activity of the aqueous extract against TA102 in the presence of S9, and of methanolic extract, with and without metabolic activation. TA100 mutagenicity was only observed for the methanolic extract in the absence of S9. The n -butanolic fraction did not present mutagenic activity. In CHO cells only the methanolic extract induced a significant increase of chromosomal aberrations in the G1/S and S phases, whereas a decrease in the mitotic index was observed in the G1/S and G2/S phases. No clastogenicity was observed for the aqueous extract. The furocoumarins (psoralen and bergapten) presented in the extracts might contribute to the mutagenicity. The lower activity of the aqueous extract was probably due to the presence of smaller amount of furocoumarins compared to the methanolic extract. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brosimum gaudichaudii Furocoumarins; Chinese hamster ovary cells; Salmonella typhimurium ; Mutagenic activity; Chromosomal aberrations
1. Introduction Drugs obtained from plants have been investigated for the possible presence of mutagenic and/or carcinogenic substances, following the criteria and norms established for synthetic medicines. The detection and evaluation of the cytotoxic, mutagenic and carcinogenic effects of plant compounds are of fundamental importance in order to reduce the possible risks of these agents. A very small portion of natural carcinogenics has been tested, but many of the plants eventually consumed by the population can have toxicant effects on a medium- or long-term basis (Gold, 1987).
* Corresponding author. Tel.: /55-16-232-0200; fax: /55-16-2220073. E-mail address: [email protected] (E.A. Varanda).
In this respect, a mention can be made of the studies conducted on the extracts of the medicinal plants Myrciaria tenella Berg. (Myrtaceae), Smilax campestris Griseb. (Smilaceae), Tripodanthus acutifoliu s Tiegh. (Loranthaceae) and Cassia corymbosa Benth. (Leguminosae), used in folk medicine in the south of Brazil, all of which presented mutagenic activity when evaluated in assays with Salmonella typhimurium . The observed mutagenicity was attributed to the presence of flavonoids, tannins and antraquinones in those extracts (Ferreira and Vargas, 1999). Although there is no direct evidence that exposure to chemical agents is responsible for genetic modifications, many experiments have shown that they can produce chromosomal aberrations and genic mutations when the mutagenic agent interacts with DNA, promoting changes in its structure that can affect the fidelity of
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 8 9 - 2
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the message and lead to irreversible changes in the cell. According to Yoshikawa et al. (1996), the recent discovery that many mutations are related to the carcinogenesis process has led to more detailed studies on mutagenesis. We investigated the mutagenic activity of Brosimum gaudichaudii Trecul (Moraceae), a widely used medicinal plant in Brazil, in Salmonella/microsome assays and chromosomal aberrations assay in Chinese hamster ovary (CHO) cells. Pozetti (1969) isolated psoralen and bergapten from B. gaudichaudii roots which are well known for their photosensitize activities (BenHur and Song, 1984). This plant is used in several ways by the Brazilian populations for vitiligo treatment: the root bark is used as a tea (Vilegas and Pozetti, 1993), in the form of baths, and the juice of the triturated root as a component added to ointments and lotions (Scavone and Panizza, 1980). The fruits are used as a kind of chewing gum (Rizzini and Mors, 1976).
2. Materials and methods 2.1. Chemicals Dimethyl sulfoxide (DMSO */CAS no. 67-68-5), nicotinamide adenine dinucleotide phosphate sodium salt (NADP */CAS no. 11-84-16-3), D-glucose-6-phosphate disodium salt (CAS no. 3671-99-6), L-histidine monohydrate (CAS no. 7048-02-4), and D-biotin (CAS no. 58-85-5) were purchased from Sigma Chemical Co. (St. Louis, MO). Standard mutagens: daunomycin (DAU*/CAS no. 23541-50-6), sodium azide (AZS, NaN3 */CAS no. 26628-22-8), 2-aminofluorene (AAF */CAS no. 153-786), 2-anthramine (2-AA */CAS no. 613-13-8), and 4 nitro-o -phenylenediamine (NPD */CAS no. 99-56-9) were also obtained from Sigma. Oxoid Nutrient Broth No. 2 (Oxoid, England) and Difco Bacto Agar (Difco, USA) were used for the preparation of bacterial growth media. All other reagents used to prepare buffers and media were from Merck (Whitehouse Station, NJ) and Sigma. The S9 fraction from Aroclor 1254-treated rats was obtained from Molecular Toxicology, Inc. (Annapolis, MD). 2.2. Mutagenicity assay Mutagenicity tests were carried out using the Salmonella/microsome assay, based on the plate-incorporation procedure, using S. typhimurium tester strains TA97a, TA98, TA100 and TA102 with and without metabolic activation (S9 mix fraction) (kindly provided by Dr B.N. Ames, Berkeley, CA). The tester strains from frozen cultures were cultured overnight in Oxoid Nutrient Broth No. 2 for 12/14 h. Mutagenicity assays were
performed using the method of Maron and Ames (1983). Different concentrations (3.37 /27 mg/plate) of aqueous or methanolic extracts and n-butanolic fraction dissolved in DMSO were added to 2 ml of top agar and 100 ml of bacterial culture and then poured onto a plate containing minimum agar. The plates were incubated at 37 8C for 48 h and the his/ colonies were manually counted. The influence of metabolic activation was tested by adding 500 ml of S9 mixture prepared with the S9 fraction obtained from liver of Sprague/Dawley rats pretreated with a polychlorinated biphenyl mixture (Aroclor 1254). All experiments were analyzed in triplicate with at least two replicates. The sample was considered to be mutagenic when the number of revertant colonies was at least twice the negative control yield (MUI E/2) and showed a significant response in analysis of variance (P 0/0.05). When only one of these criteria was met, the compound was considered to present signs of positive response, in agreement with McGeorge et al. (1983) and Vargas et al. (1993). The standard mutagens used as positive controls in each experiment were 2-anthramine (0.125 mg/plate) and sodium azide (1.25 mg/plate) for TA100, 4 nitro-o phenylenediamine (5 mg/plate) and 2-anthramine (0.125 mg/plate) for TA98 and TA97a, and daunomycin (3 mg/ plate) and 2-aminofluorene (10 mg/plate) for TA102. 2.3. Chromosome aberrations in CHO cells CHO9 cells were maintained as monolayers growing at 37 8C in 25 cm2 plastic flasks (Corning) containing HAM-F10 (Sigma) plus DEM (Dulbecco’s Modified Eagle’s Medium-Sigma), supplemented with 10% fetal serum (Cultilab), antibiotics (0.01 mg/ml streptomycin and 0.005 mg/ml penicillin) and 2.38 mg/ml Hepes (Sigma). Cells were subcultured two or three times a week, washed in phosphate buffered saline and treated with ATV (0.2% trypsin and 0.02% versene */Institute Adolfo Lutz) for removal from the inner surface of the cultured tube. The methanolic extract was diluted in DMSO and used at a final concentration of 0.4, 0.8 and 1.2 mg/ml in culture. Similarly the aqueous extract was used at final concentrations of 0.8, 1.2 and 1.6 mg/ml in culture. The final volume of DMSO used in solvent control cultures was 40 ml/culture. Cells were seeded at a density of 106 cells/flask and then treated with methanolic or aqueous Brosimum extracts in different times during the cell cycle, and remained in the culture until cell harvesting. Exponentially growing CHO cells have a doubling time of 12/14 h (Preston et al., 1987; Cozzi et al., 1993). In this work, total culture time was 12 h. Cells were harvested 3, 8 and 12 h after initiation of treatment. According to Preston et al. (1981), cells fixed 3, 8 and 12 h after the beginning of treatment are, respectively, in the late G2/S, middle S
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and early G1/S phases of the cell cycle at the moment they are treated. Each experiment was carried out in three replicates (control, solvent control and treated cultures). Colcemid (Sigma) at a final concentration of 0.1 mg/ml was added to the culture medium 2 h before harvesting. The cells were harvested by the method of Moorhead et al. (1960), adapted for CHO cells (1% sodium citrate as hypotonic solution and 3:1 methanol/acetic acid as fixative). The air-dried chromosome preparations were stained with 3% Giemsa diluted in phosphate buffer. Three hundred metaphases per treatment were analyzed to determine the frequency of chromosome aberrations. The mitotic index was obtained by counting the number of mitotic cells for a total of 6000 cells analyzed per treatment. The differences in the frequency of chromosomal aberrations and in mitotic index between cells treated with Brosimum extracts and controls were analyzed statistically by analysis of variance, with calculations of the F -statistic and respective P values. When P B/ 0.05, the mean values of each treatment were compared by the Turkey test, in which the calculation of the minimum significant difference for P is 0.05.
259
the solvent was added to 200 ml of the aqueous phase in a separatory funnel and the mixture was shaken and left to stand until two phases were formed. The n-butanolic fraction (upper) was collected and the process repeated two times. The n -butanolic fractions were gathered, the solvent was evaporated and the dry residue was weighed and dissolved in DMSO to obtain a solution containing 0.135 g/ml DMSO. 2.5. HPLC analysis of the extracts The samples were submitted to High Performance Liquid Chromatography (HPLC) using a Shimadzu LC10 AD chromatograph equipped with a photodiode array detector and a Supelcosil C-18 column (4.6 /25 cm) with 5 mm particles. The furocoumarins psoralen and bergapten were determined with the use of authentic standards from a collection of our laboratory. MeOH:H2O was used as the mobile phase in a linear gradient in proportions of 40:60 0/80:20 in 20 min, staying for 5 min at these conditions. The sample volume injected was 20 ml.
3. Results 2.4. Extract preparation 3.1. Mutagenicity results Plant sample (root) of B. gaudichaudii was collected in Araraquara, Sa˜o Paulo-Brazil and was taxonomically identified by the authors using identified specimens and floristic reference. A voucher specimen is kept at Herba´rio Alberto Castellanos (GUA) */Rio de Janeiro (GUA 44782). The root bark was dried in an oven with forced air circulation at 50 8C for 24 h and powdered in a mill. The aqueous extract was obtained from 5 g of the root bark powder added to 250 ml of water and submitted to ultrasound waves for 30 min. The material was then filtered and the procedure was repeated twice. The aqueous extract was lyophilized, and the dry residue dissolved in DMSO to obtain a solution containing 0.135 g/ml of DMSO. The dried powder (5 g) was placed in 250 ml methanol, submitted to ultrasound waves for 30 min, and filtered. The procedure was repeated twice. After weighing, the dry extract was dissolved in DMSO at the concentration of 0.135 g/ml. A 1% solution of the root bark powder in distilled water was submitted to ultrasound waves for 30 min, filtered and partitioned with chloroform. In a separatory funnel, chloroform (50 ml) was added to the aqueous extract (200 ml), and the mixture was shaken and left to stand until two phases formed. The chloroform phase (lower) was collected and the process was repeated two times. The aqueous phase (upper) was submitted to partition with n -butanol. For this procedure, 50 ml of
The mutagenic activity data obtained in the Salmonella/microsome assay are shown in Table 1. The table shows the presence of mutagenicity induction for the aqueous extract in the presence of S9 and signs of mutagenicity in the absence of S9 for the TA102 strain, and signs of mutagenicity for TA100 in the presence of S9. The methanolic extract induced mutagenicity in TA102 (/S9 and /S9) and in TA100 (in the absence of S9, and signs of mutagenicity in the presence of S9). Higher mutagenic indexes (namely, ratio between the average of the revertant number induced per plate for each concentration and the average of the number of revertants of the negative control) were obtained in the presence of metabolic activation in TA102, which indicate the presence of oxidative mutagens (Table 2). Table 1 also shows that the methanolic extract at the concentration of 13.5 mg/plate presented signs of toxicity for TA100 and TA97a. Concentrations above 20.25 mg/plate were toxic for all strains because, the revertant frequencies were below the normal spontaneous frequency. Mutagenicity was not observed in any strain when the n-butanolic fraction was evaluated in the presence or absence of S9. 3.2. Chromosomal aberrations The results obtained through analysis of chromosomal aberrations and mitotic index for CHO cells treated
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Table 1 Mutagenic activity of aqueous and methanolic extracts and of the n -butanolic fraction from B. gaudichaudii on S. typhimurium strains Extract/fraction
Dose mg/plate
Revertants his/plate (mean9S.D.)a TA102
TA100
TA98
TA97a
S9
S9
S9
S9
S9
S9
S9
S9
Aqueous extract
3.37 6.75 10.12 13.50 20.25 27.00
255962 369953 313933 388946* 407914** 336956
521997 540935** 644920** 7289102** 10209100** 9659378
188921 178933 / 196913 191905 199927
227938 213908* 215907* 273963 331966* 298957*
30907 34906 / 39906 39909 45910
55908 61904 61901 66917 54921 41904
159928 151930 / 156935 165941 155933
/ 228922 / 292926 283961 210962
Methanolic extract
3.37 6.75 10.12 13.50 20.25
6019214 418975 9309339* 5069158 /
1073990** 1353981** 7189275 8409180* /
430947** 417995* 341954* 64933 25924
281998 283913** 269915* 105911 /
49924 50936 47923 47933 2902
58916 40908 50932 36908 /
154914 187974 17916 / /
167962 133930 49927 10902 /
n -Butanolic fraction
3.37 6.75 10.12 13.50 20.25
249940 277913 287932 315933 228928
300922 314941 343919 353942 330925
175913 196905 176907 192909 149915
148903 130914 166924 154916 172916
27905 29904 29904 23903 19902
69903 54908 / 54905 52907
185907 179924 176920 171915 180914
191914 194914 198916 183920 217938
Negative
Control
276921
321922
179924
173906
36908
41905
157914
169928
Positive
Control
13219104
1015985
11179255
11049125
13289182
333939
1038982
11879110
Negative control 0.1 ml DMSO (extract solvent). Positive control 2-anthramine (0.625 mg/plate)0 TA100 and TA98 (S9); 2-aminofluorene (10 mg/plate)0 TA102 and TA97a (S9); daunomycin (3 mg/plate)0 TA102 (S9); sodium azide (1.25 mg/plate)0 TA100 (S9); 4-nitro-o phenylenediamine (5 mg/plate)0 TA98 and TA97a (S9). a Values represent the mean9S.D. of at least two experiments carried out in triplicate. * P B 0.05 (ANOVA). ** P B 0.01 (ANOVA). Table 2 Mutagenic indexes for the aqueous and methanolic extracts and for the n -butanolic fraction from B. gaudichaudii in S. typhimurium strains Treatment mg/plate
Mutagenic indexesa TA102
TA100
TA98
TA97a
S9
S9
S9
S9
S9
S9
S9
S9
Aqueous extracts 3.37 6.75 10.12 13.50 20.25 27.00
0.92 1.33 1.13 1.41 1.47 1.22
1.62 1.68 2.00 2.26 3.18 3.01
1.05 0.99 / 1.09 1.07 1.11
1.31 1.23 1.24 1.58 1.91 1.72
0.83 0.94 / 1.08 1.08 1.25
1.37 1.34 1.49 1.61 1.32 1.00
1.01 0.96 / 0.99 1.05 0.92
/ 1.35 / 1.24 1.67 1.24
Methanolic extract 3.37 6.75 10.12 13.50 20.25
2.18 1.51 3.37 1.83 /
3.34 4.21 2.24 2.62 /
2.40 2.33 1.91 0.36 0.14
1.62 1.64 1.55 0.61 /
1.36 1.39 1.31 1.31 0.05
1.41 0.98 1.22 0.88 /
0.98 1.19 0.11 / /
0.99 0.79 0.29 0.06 /
n-Butanolic fraction 3.37 6.75 10.12 13.50 20.25
0.90 1.00 1.03 1.14 0.82
1.07 0.98 1.07 1.10 1.03
0.98 1.09 0.98 1.07 0.83
0.86 0.75 0.96 0.89 0.99
0.75 0.80 0.80 0.64 0.53
1.68 1.32 / 1.32 1.27
1.18 1.14 1.12 1.09 1.15
1.13 1.15 1.17 1.08 1.28
a
Mutagenic index (MUI): number of his (revertant colonies) induced in the sample/number of spontaneous his in the negative control. Mutagenic index in bold type indicates that the number of revertants is twice of the negative controls yields.
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261
Table 3 Distribution of the different types of chromosomal aberrations and mitotic index observed in CHO cells after treatment with methanolic extract of B. gaudichaudii in different times (12, 8 and 3 h) during the cell cycle Treatment (mg/ml)
Chromosomal aberrations Gap
Break
Total
NCA
MI (%)
OTA
c
ic
c
ic
12 h (G1 /S) Control DMSO 0.4 0.8 1.2
21 18 62 46 63
5 5 13 5 7
7 3 3 7 39
/ / / 1 12
1d 1t, 1d 2t, 3d 1t 14t, 1d
34 28 83* 60* 136*
32 36 67* 53* 92*
4.50 4.00 5.10 4.40 2.00*
8 h (S phase) Control DMSO 0.4 0.8 1.2
13 21 12 21 24
4 3 4 3 10
6 2 20 45 40
3 1 5 5 7
/ 1t, 1d / 7t 16t, 1d
26 29 41* 81* 98*
24 28 34* 73* 98*
6.00 5.00 5.30 6.50 4.40
3 h (G2 /S) Control DMSO 0.4 0.8 1.2
9 24 19 9 27
/ / 1 12 /
12 6 9 39 34
/ 1 4 5 1
/ 2f, 3t 2t 3t, 1q 5t, 2f, 2q
21 36 35 69 71
21 31 32 60 60
6.70 7.80 5.60* 4.30* 2.10*
Three hundred cells were analyzed per treatment; c, chromatid-type; ic, isochromatid-type; d, dicentric; f, fragments; q, quadriradial; t, triradial; NCA, number of cells with aberrations; MI, mitotic index; OTA, other types of aberrations. * Statistically different when compared with solvent control.
Table 4 Distribution of the different types of chromosomal aberrations and mitotic index observed in CHO cells after treatment with aqueous extract of B. gaudichaudii in different times (12, 8 and 3 h) during the cell cycle Treatment (mg/l)
Chromosomal aberrations Gap
Break c
Total
NCA
MI (%)
OTA
c
ic
ic
12 h (G1 /S) Control DMSO 1.2 1.4 1.6
15 16 29 26 16
3 2 3 6 3
6 8 5 2 /
1 / 5 1 1
/ 1t 1t 1q 3t, 1r, 1d
25 27 43 36 25
25 26 35 35 22
5.33 5.53 4.87 4.10 5.53
8 h (S) Control DMSO 1.2 1.4 1.6
7 6 16 29 18
5 1 3 1 6
16 2 11 4 15
3 / 3 4 4
1d / 3t, 1q 2r, 2d, 3t 2d, 2t
32 9 37 45 47
29 9 35 37 44
6.63 5.87 5.80 4.40 6.00
3 h (G2 /S) Control DMSO 1.2 1.4 1.6
8 11 9 12 28
1 3 2 / 4
5 16 11 6 11
/ 1 1 / 1
1q / 1t / 2d, 4t, 1q
15 31 24 18 56
15 29 22 17 54
6.00 6.30 5.03 4.40 3.23
Three hundred cells were analyzed per treatment; c, chromatid-type; ic, isochromatid-type; d, dicentric; q, quadriradial; t, triradial; r, ring; NCA, number of cells with aberrations; MI, mitotic index; OTA, other types of aberrations.
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in different times during the cell cycle are shown in Tables 3 and 4. Treatment with the various concentrations (1.2 /1.6 mg/ml) of the aqueous extract of Brosimum (Table 4) caused no significant increase in the number of chromosomal aberrations, in the frequency of cells with aberrations or in mitotic index. In contrast, treatment with the methanolic extract (Table 3) at all concentrations used (0.4 /1.2 mg/ml) caused a significant increase (P B/ 0.05) in the number of cells with chromosomal aberrations and in the frequency of such cells when they were treated in the G1/S and S phases. The mitotic index decreased significantly with increasing methanolic extract concentrations in the G2/S phase, and in the G1/S phase when the highest concentration was used. The most frequent aberrations were chromatid gaps and breaks. Other types of aberrations such as isochromatid gaps and breaks, quadriradial and triradial figures, dicentrics and fragments were also observed. When statistical analysis was conducted without considering gaps, the results obtained were the same.
4. Discussion and conclusions The methanolic extract of the plant induced a significant increase in the number of revertants for the TA100 strain in the absence of S9, and signs of mutagenicity in the presence of S9, and for TA102, in the presence and absence of S9. The aqueous extract only induced a significant increase in number of revertants in TA102 and signs of mutagenicity in TA100 in the presence of S9. The n-butanolic fraction did not induce mutagenicity. The results clearly showed an increase in chromosomal aberrations in the cultures treated with the methanolic extract in the G1/S and S phases of the cell cycle. In the G2/S phase an increase also occurred in the number of aberrations and in the frequency of cells with aberrations, which, however, was not significant. The mitotic index was significantly reduced in cultures treated in the G1/S phase with 1.2 mg/ml concentration, and in cultures treated in the G2/S phase with all
concentrations tested. Table 5 summarizes the results on the genotoxicity of B. gaudichaudii . The psoralens are tricyclic compounds consisting of a furan ring fused with a coumarin. According to Lagatolla et al. (1998), they cause countless biological effects, but the more commonly accepted one is the effect on DNA. The psoralens are secondary plant metabolites found in many fruits and vegetables. Synthetic forms of 5methoxypsoralen (bergapten) and 8-methoxypsoralen (8-MOP) have been used in combination with UV radiation in skin photochemotherapy for decades. However, handling or ingestion of psoralen-containing plants as well as medicinal use of these compounds has been shown to cause human health hazards (dermatitis, hepatotoxicity) (Diawara et al., 2000). The reaction of 8-MOP with DNA starts by a process of intercalation of the chemical compound between the stacked bases of DNA. In the absence of light, no covalent binding is established between the two molecules. However, absorption of near-UV light (320 /400 nm) allows 8-MOP to react either through its furan or pyrone moiety with a pyrimidine (preferably thymine) in one DNA strand, yielding monoadducts. Further absorption of light in the same wavelength range allows the reaction of some of the monoadducts */those already formed by the furan side of the psoralen*/ with another pyrimidine placed in an adjacent position on the opposite DNA strand, thus, yielding interstrand cross-links (BenHur and Song, 1984; Cimino et al., 1985) Psoralens are agents that intercalate in the DNA molecule forming mono- and di-adducts, and they also induce the production of singlet oxygen and superoxide radicals (Pathak and Joshy, 1984; Bianchi et al., 1996) that are oxidative mutagens clearly detected by the TA102 strain. This strain has an A /T base-pair at the critical site for reversion; it detects a variety of oxidative mutagens, active forms of oxygen, and has an intact excision-repair pathway (Levin et al., 1982). Furocoumarins appear to be able to generate 1O2 even when complexed with DNA (DeMol et al., 1981). The
Table 5 Summary of results on the genotoxicity of B. gaudichaudii Extract/fraction
Salmonella assaysa TA102
Aqueous extract Methanolic extract n -Butanolic fraction
CHO assays
TA100
TA98
TA97a
CA
MI
S9
S9
S9
S9
S9
S9
S9
S9
G1/S
S
G2/S
G1/S
S
G2/S
9
9 9
nt
nt
nt
nt
nt
nt
, Positive (ANOVA, P 0 0.05; MUIE 2); , negative; 9, signs of positive response (ANOVA, P B 0.05; MUIB 2); nt, not tested; CA, chromosomal aberrations; MI, mitotic index; G1/S, S, G2/S, cell cycles phases. a S9, without metabolic activation; S9, with metabolic activation.
E.A. Varanda et al. / Journal of Ethnopharmacology 81 (2002) 257 /264
mutagenic effect of 1O2 may even be enhanced in the immediate proximity of DNA because of its longer life time in the hydrophobic DNA regions (Merkel and Kearns, 1972; Ito, 1980). Furthermore, 1O2 formed outside DNA by free furocoumarins in the cytoplasm or by furocoumarins noncovalently bound to proteins may contribute to the genetic effects, because diffusion of 1O2 over relatively large portions of the cells seems possible (Pooler and Valenzeno, 1979; Ito, 1980). Gamper et al. (1987) and Boyer et al. (1988) showed that psoralens react preferentially with thymidine residues, with interstrand cross-link formation strongly favored at 5?-TpA-3? sites. A 5?-TpA-3? site exists adjacent to the hisG46 codon (Hartman et al., 1986) of S. typhimurium strain TA100. According to Levin and Ames (1986) and Koch (1986), psoralen induced base-pair substitution in Salmonella . In addition, we observed mutagenic activity in the TA100 Salmonella strain which detects mutagens that cause base-pair substitutions (Maron and Ames, 1983). Considering that furocoumarins, bergapten and psoralen, are the major components (60%) of the methanolic extract of B. gaudichaudii , it can be inferred that both the mutagenicity and clastogenicity observed might have been induced by these agents. The result obtained with the aqueous extract was less marked, probably because in the process of aqueous extraction the amount of furocoumarins (3%) removed from the plant was much smaller than that obtained by methanol extraction. This was probably the reason why the increase in the chromosomal aberrations in CHO cells exposed to the aqueous extract was not significant and was also less marked in the assays with Salmonella . HPLC analysis (data not shown) revealed that the n butanol fraction contained only traces of furocoumarins. This was expected, since furocoumarins were previously removed by the partition with chloroform. The absence of mutagenicity in the n -butanol fraction thus reinforces the hypothesis that furocoumarins are the components responsible for the mutagenicity. In fact, the literature reports a remarkable mutagenicity (Bianchi et al., 1996; Cebula and Koch, 1990) as well as toxicity (Ivie, 1987) of furocoumarins. Even though the culture dishes and bottles for the bacteria were incubated in the dark, all the experiments were performed in the presence of natural light, a fact that might have allowed the photoactivation of the furocoumarins. As observed in the present study, extracts of the root bark of B. gaudichaudii showed mutagenic activity in the S. typhimurium test and on CHO cells. Thus, caution should be taken in using these roots for the treatment of vitiligo in popular medicine. Additional studies are needed to identify other possible substances that might contribute to the effects observed.
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Acknowledgements The authors greatly thank FAPESP, CNPq and PADC-FCF-UNESP for their financial support and Prof. A.T. Natarajan (University of Leiden, The Netherlands) that supplied CHO cells (CHO9 line).
References BenHur, E., Song, P.S. 1984. The photochemistry and photobiology of furocoumarins (psoralens). Advances in Radiation Bioliology 11, 131 /171. Bianchi, L., Melli, R., Pizzala, R., Stivala, L.A., Rehak, L., Quarta, S., Vannini, V. 1996. Effects of b-carotene and a-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen. Mutation Research 369, 183 /194. Boyer, V., Moustacchi, E., Sage, E. 1988. Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity. Biochemistry 27, 3011 /3018. Cebula, T.A., Koch, W.H. 1990. Analysis of spontaneous and psoralen-induced Salmonella typhimurium his G46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences. Mutation Research 229, 79 / 87. Cimino, G.D., Gamper, H.B., Isaacs, S.T., Hearst, J.E. 1985. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annual Review of Biochemistry 54, 1151 /1193. Cozzi, R., Nicolai, M., Perticone, P., De Salvia, R., Spuntarelli, F. 1993. Desmutagenic activity of natural humic acids: inhibition of mitomycin C and maleic hydrazide mutagenicity. Mutation Research 299, 37 /44. DeMol, N.J., Beijersbegen van Henegouwen, G.M.I., Mohn, G.R., Glickman, B.W., van Kleef, P.M. 1981. On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UV-A irradiation in Escherichia coli K-12. Mutation Research 82, 23 /30. Diawara, M.M., Williams, D.E., Oganesian, A., Spitsbergen, J. 2000. Dietary psoralens induce hepatotoxicity in C57 mice. Journal of Natural Toxins 9, 179 /195. Ferreira, I.C.D.F., Vargas, V.M.F. 1999. Mutagenicity of medicinal plant extracts in Salmonella /microssome assay. Phytotherapy Research 13, 397 /400. Gamper, H.B., Cimino, G.D., Hearst, J.E. 1987. Solution hybridization of cross-linkable DNA oligonucleotides to bacteriophage M13 DNA: effect of secondary structure on hybridization kinetics and equilibria. Journal of Molecular Biology 197, 349 /362. Gold, L.S. 1987. Second Chronological Supplement to the Carcinogenic Potency Database: Standardized Results of Animal Bioassays Published Through December 1984 and by the National Toxicology Program through May 1986. Environmental Health Perspective, Triangle Park, p. 7. Hartman, P.E., Ames, B.N., Roth, J.R., Barnes, W.M., Levin, D.E. 1986. Target sequences for mutagenesis in Salmonella histidinerequiring mutants. Environmental Mutagenesis 8, 631 /641. Ito, T. 1980. The dependence of photo-sensitizing efficacy of acridine orange and toluidine blue on the degree of sensitizer-cell interaction. Photochemistry Photobiology 31, 565 /570. Ivie, G.W. 1987. The chemistry of plant furocoumarins and their medical, toxicological, environmental and coevolutionary significance. Revista Latinoamericana de Quimica 18, 1 /6. Koch, W.H. 1986. Psoralen photomutagenic specificity in Salmonella typhimurium . Mutation Research 160, 195 /205.
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Lagatolla, C., Dolzani, L., Granzotto, M., Monti-Bragadin, C. 1998. Genotoxic activity of 4,4?,5?-trimethylazapsoralen on plasmid DNA. Teratogenesis Carcinogenesis and Mutagenesis 18, 239 /248. Levin, D.E., Ames, B.N. 1986. Classifying mutagens as to their specificity in causing the six possible transition and transversions: a simple analysis using the Salmonella mutagenicity assay. Environmental Mutagenesis 8, 9 /28. Levin, D.E., Hollsteins, M., Christman, M.F., Schwiers, E.A., Ames, B.N. 1982. A new Salmonella tester strain (TA102) with A /T base pair at the site of mutation detects oxidative mutagens. Proceedings of the National Academy of Science USA 79, 7445 /7449. Maron, D.M., Ames, S.N. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173 /215. McGeorge, L.J., Louis, J.B., Atherholt, T.B., McGarrit, G.J., 1983. Mutagenicity analysis of industrial effluents: background, results to date. Report of the New Jersey Department of Environmental Protection, Trenton, appendix G. Merkel, P.B., Kearns, D.R. 1972. Radiationless decay of singlet molecular oxygen in solution. An experimental and theoretical study of electronic and vibrational energy transfer. Journal of the American Chemical Society 94, 7244 /7253. Moorhead, P.S., Nowell, P.C., Mellman, W.L., Battipps, M.P., Hungerfor, D.A. 1960. Chromosome preparation of leukocytes cultured from human peripheral blood. Experimental Cell Research 20, 613 /616. Pathak, M.A., Joshy, P.A. 1984. Production of active oxygen species (1O2 and O2 ) by psoralen and ultraviolet radiation (320 /400 nm). Biochimica Biophysica Acta 798, 115 /126.
Pooler, J.P., Valenzeno, D.P. 1979. The role of singlet oxygen in photooxidation of excitable cell membranes. Photochemistry and Photobiology 30, 581 /584. Pozetti, G.L. 1969. Contribuic¸a˜o ao estudo quı´mico do Brosimum gaudichaudii Tre´cul.1.Isolamento e identificac¸a˜o do bergapteno e do psoraleno das raı´zes do Brosimum gaudichaudii Tre´cul. Revista Faculdade de Farma´cia e Odontologia de Araraquara 3, 215 /223. Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., MacFee, A.F., Wolff, S., Wasson, J.S. 1981. Mammalian in vivo and in vitro cytogenetic assays, a report of the EPA’s Gene-Tox-Program. Mutation Research 87, 143 / 188. Preston, R.J., San Sebastian, J.R., MacFee, A.F. 1987. The in vitro human lymphocyte assay for assessing the clastogenicity of chemical agents. Mutation Research 189, 175 /183. Rizzini, C.T., Mors, W.B. 1976. Botaˆnica Econoˆmica Brasileira. FPUEDUSP, Sa˜o Paulo, p. 12. Scavone, O., Panizza, S. 1980. Plantas To´xicas. Codec-UPS, Sa˜o Paulo, p. 89. Vargas, V.M.F., Motta, V.E.P., Henriques, J.A.P. 1993. Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries. Mutation Research 319, 31 /45. Vilegas, W., Pozetti, G.L. 1993. Coumarins from Brosimum gaudichaudii . Journal of Natural Products 56, 416 /417. Yoshikawa, K., Inagaki, K., Terashita, J., Shishiyama, T., Kuo, S., Shankel, D.M. 1996. Antimutagenic activity of extracts from Japanese eggplant. Mutation Research 371, 65 /71.
Genotoxicity of Brosimum gaudichaudii measured by the Salmonella/ microsome assay and chromosomal aberrations in CHO cells Eliana Aparecida Varanda a,*, Gilberto Luiz Pozetti b, Miriam Verginia Lourenc¸o b, Wagner Vilegas b, Maria Stella Gonc¸alves Raddi a a
Departamento de Cieˆncias Biolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas de Araraquara, Universidade Estadual Paulista, UNESP, Rodovia Araraquara Jau´-Km 1, 14801-902 Araraquara, SP, Brazil b Instituto de Quı´mica de Araraquara, Universidade Estadual Paulista, UNESP, Sa˜o Paulo, Brasil. Rua Prof. Francisco Degni s/n, 14801-970 Araraquara, SP, Brazil Received 31 May 2001; received in revised form 31 March 2002; accepted 4 April 2002
Abstract The root bark of Brosimum gaudichaudii Tre´cul (Moraceae) is popularly used for treatment of vitiligo. In the present study the mutagenic activity of the aqueous and methanolic extract as well as of the n -butanolic fraction of this medicinal plant were evaluated using Salmonella typhimurium assays, TA100, TA98, TA102 and TA97a strains, while the clastogenic effect in Chinese hamster ovary (CHO) cells in the G1/S, S and G2/S phases of the cell cycle. The results showed mutagenic activity of the aqueous extract against TA102 in the presence of S9, and of methanolic extract, with and without metabolic activation. TA100 mutagenicity was only observed for the methanolic extract in the absence of S9. The n -butanolic fraction did not present mutagenic activity. In CHO cells only the methanolic extract induced a significant increase of chromosomal aberrations in the G1/S and S phases, whereas a decrease in the mitotic index was observed in the G1/S and G2/S phases. No clastogenicity was observed for the aqueous extract. The furocoumarins (psoralen and bergapten) presented in the extracts might contribute to the mutagenicity. The lower activity of the aqueous extract was probably due to the presence of smaller amount of furocoumarins compared to the methanolic extract. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brosimum gaudichaudii Furocoumarins; Chinese hamster ovary cells; Salmonella typhimurium ; Mutagenic activity; Chromosomal aberrations
1. Introduction Drugs obtained from plants have been investigated for the possible presence of mutagenic and/or carcinogenic substances, following the criteria and norms established for synthetic medicines. The detection and evaluation of the cytotoxic, mutagenic and carcinogenic effects of plant compounds are of fundamental importance in order to reduce the possible risks of these agents. A very small portion of natural carcinogenics has been tested, but many of the plants eventually consumed by the population can have toxicant effects on a medium- or long-term basis (Gold, 1987).
* Corresponding author. Tel.: /55-16-232-0200; fax: /55-16-2220073. E-mail address: [email protected] (E.A. Varanda).
In this respect, a mention can be made of the studies conducted on the extracts of the medicinal plants Myrciaria tenella Berg. (Myrtaceae), Smilax campestris Griseb. (Smilaceae), Tripodanthus acutifoliu s Tiegh. (Loranthaceae) and Cassia corymbosa Benth. (Leguminosae), used in folk medicine in the south of Brazil, all of which presented mutagenic activity when evaluated in assays with Salmonella typhimurium . The observed mutagenicity was attributed to the presence of flavonoids, tannins and antraquinones in those extracts (Ferreira and Vargas, 1999). Although there is no direct evidence that exposure to chemical agents is responsible for genetic modifications, many experiments have shown that they can produce chromosomal aberrations and genic mutations when the mutagenic agent interacts with DNA, promoting changes in its structure that can affect the fidelity of
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 8 9 - 2
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the message and lead to irreversible changes in the cell. According to Yoshikawa et al. (1996), the recent discovery that many mutations are related to the carcinogenesis process has led to more detailed studies on mutagenesis. We investigated the mutagenic activity of Brosimum gaudichaudii Trecul (Moraceae), a widely used medicinal plant in Brazil, in Salmonella/microsome assays and chromosomal aberrations assay in Chinese hamster ovary (CHO) cells. Pozetti (1969) isolated psoralen and bergapten from B. gaudichaudii roots which are well known for their photosensitize activities (BenHur and Song, 1984). This plant is used in several ways by the Brazilian populations for vitiligo treatment: the root bark is used as a tea (Vilegas and Pozetti, 1993), in the form of baths, and the juice of the triturated root as a component added to ointments and lotions (Scavone and Panizza, 1980). The fruits are used as a kind of chewing gum (Rizzini and Mors, 1976).
2. Materials and methods 2.1. Chemicals Dimethyl sulfoxide (DMSO */CAS no. 67-68-5), nicotinamide adenine dinucleotide phosphate sodium salt (NADP */CAS no. 11-84-16-3), D-glucose-6-phosphate disodium salt (CAS no. 3671-99-6), L-histidine monohydrate (CAS no. 7048-02-4), and D-biotin (CAS no. 58-85-5) were purchased from Sigma Chemical Co. (St. Louis, MO). Standard mutagens: daunomycin (DAU*/CAS no. 23541-50-6), sodium azide (AZS, NaN3 */CAS no. 26628-22-8), 2-aminofluorene (AAF */CAS no. 153-786), 2-anthramine (2-AA */CAS no. 613-13-8), and 4 nitro-o -phenylenediamine (NPD */CAS no. 99-56-9) were also obtained from Sigma. Oxoid Nutrient Broth No. 2 (Oxoid, England) and Difco Bacto Agar (Difco, USA) were used for the preparation of bacterial growth media. All other reagents used to prepare buffers and media were from Merck (Whitehouse Station, NJ) and Sigma. The S9 fraction from Aroclor 1254-treated rats was obtained from Molecular Toxicology, Inc. (Annapolis, MD). 2.2. Mutagenicity assay Mutagenicity tests were carried out using the Salmonella/microsome assay, based on the plate-incorporation procedure, using S. typhimurium tester strains TA97a, TA98, TA100 and TA102 with and without metabolic activation (S9 mix fraction) (kindly provided by Dr B.N. Ames, Berkeley, CA). The tester strains from frozen cultures were cultured overnight in Oxoid Nutrient Broth No. 2 for 12/14 h. Mutagenicity assays were
performed using the method of Maron and Ames (1983). Different concentrations (3.37 /27 mg/plate) of aqueous or methanolic extracts and n-butanolic fraction dissolved in DMSO were added to 2 ml of top agar and 100 ml of bacterial culture and then poured onto a plate containing minimum agar. The plates were incubated at 37 8C for 48 h and the his/ colonies were manually counted. The influence of metabolic activation was tested by adding 500 ml of S9 mixture prepared with the S9 fraction obtained from liver of Sprague/Dawley rats pretreated with a polychlorinated biphenyl mixture (Aroclor 1254). All experiments were analyzed in triplicate with at least two replicates. The sample was considered to be mutagenic when the number of revertant colonies was at least twice the negative control yield (MUI E/2) and showed a significant response in analysis of variance (P 0/0.05). When only one of these criteria was met, the compound was considered to present signs of positive response, in agreement with McGeorge et al. (1983) and Vargas et al. (1993). The standard mutagens used as positive controls in each experiment were 2-anthramine (0.125 mg/plate) and sodium azide (1.25 mg/plate) for TA100, 4 nitro-o phenylenediamine (5 mg/plate) and 2-anthramine (0.125 mg/plate) for TA98 and TA97a, and daunomycin (3 mg/ plate) and 2-aminofluorene (10 mg/plate) for TA102. 2.3. Chromosome aberrations in CHO cells CHO9 cells were maintained as monolayers growing at 37 8C in 25 cm2 plastic flasks (Corning) containing HAM-F10 (Sigma) plus DEM (Dulbecco’s Modified Eagle’s Medium-Sigma), supplemented with 10% fetal serum (Cultilab), antibiotics (0.01 mg/ml streptomycin and 0.005 mg/ml penicillin) and 2.38 mg/ml Hepes (Sigma). Cells were subcultured two or three times a week, washed in phosphate buffered saline and treated with ATV (0.2% trypsin and 0.02% versene */Institute Adolfo Lutz) for removal from the inner surface of the cultured tube. The methanolic extract was diluted in DMSO and used at a final concentration of 0.4, 0.8 and 1.2 mg/ml in culture. Similarly the aqueous extract was used at final concentrations of 0.8, 1.2 and 1.6 mg/ml in culture. The final volume of DMSO used in solvent control cultures was 40 ml/culture. Cells were seeded at a density of 106 cells/flask and then treated with methanolic or aqueous Brosimum extracts in different times during the cell cycle, and remained in the culture until cell harvesting. Exponentially growing CHO cells have a doubling time of 12/14 h (Preston et al., 1987; Cozzi et al., 1993). In this work, total culture time was 12 h. Cells were harvested 3, 8 and 12 h after initiation of treatment. According to Preston et al. (1981), cells fixed 3, 8 and 12 h after the beginning of treatment are, respectively, in the late G2/S, middle S
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and early G1/S phases of the cell cycle at the moment they are treated. Each experiment was carried out in three replicates (control, solvent control and treated cultures). Colcemid (Sigma) at a final concentration of 0.1 mg/ml was added to the culture medium 2 h before harvesting. The cells were harvested by the method of Moorhead et al. (1960), adapted for CHO cells (1% sodium citrate as hypotonic solution and 3:1 methanol/acetic acid as fixative). The air-dried chromosome preparations were stained with 3% Giemsa diluted in phosphate buffer. Three hundred metaphases per treatment were analyzed to determine the frequency of chromosome aberrations. The mitotic index was obtained by counting the number of mitotic cells for a total of 6000 cells analyzed per treatment. The differences in the frequency of chromosomal aberrations and in mitotic index between cells treated with Brosimum extracts and controls were analyzed statistically by analysis of variance, with calculations of the F -statistic and respective P values. When P B/ 0.05, the mean values of each treatment were compared by the Turkey test, in which the calculation of the minimum significant difference for P is 0.05.
259
the solvent was added to 200 ml of the aqueous phase in a separatory funnel and the mixture was shaken and left to stand until two phases were formed. The n-butanolic fraction (upper) was collected and the process repeated two times. The n -butanolic fractions were gathered, the solvent was evaporated and the dry residue was weighed and dissolved in DMSO to obtain a solution containing 0.135 g/ml DMSO. 2.5. HPLC analysis of the extracts The samples were submitted to High Performance Liquid Chromatography (HPLC) using a Shimadzu LC10 AD chromatograph equipped with a photodiode array detector and a Supelcosil C-18 column (4.6 /25 cm) with 5 mm particles. The furocoumarins psoralen and bergapten were determined with the use of authentic standards from a collection of our laboratory. MeOH:H2O was used as the mobile phase in a linear gradient in proportions of 40:60 0/80:20 in 20 min, staying for 5 min at these conditions. The sample volume injected was 20 ml.
3. Results 2.4. Extract preparation 3.1. Mutagenicity results Plant sample (root) of B. gaudichaudii was collected in Araraquara, Sa˜o Paulo-Brazil and was taxonomically identified by the authors using identified specimens and floristic reference. A voucher specimen is kept at Herba´rio Alberto Castellanos (GUA) */Rio de Janeiro (GUA 44782). The root bark was dried in an oven with forced air circulation at 50 8C for 24 h and powdered in a mill. The aqueous extract was obtained from 5 g of the root bark powder added to 250 ml of water and submitted to ultrasound waves for 30 min. The material was then filtered and the procedure was repeated twice. The aqueous extract was lyophilized, and the dry residue dissolved in DMSO to obtain a solution containing 0.135 g/ml of DMSO. The dried powder (5 g) was placed in 250 ml methanol, submitted to ultrasound waves for 30 min, and filtered. The procedure was repeated twice. After weighing, the dry extract was dissolved in DMSO at the concentration of 0.135 g/ml. A 1% solution of the root bark powder in distilled water was submitted to ultrasound waves for 30 min, filtered and partitioned with chloroform. In a separatory funnel, chloroform (50 ml) was added to the aqueous extract (200 ml), and the mixture was shaken and left to stand until two phases formed. The chloroform phase (lower) was collected and the process was repeated two times. The aqueous phase (upper) was submitted to partition with n -butanol. For this procedure, 50 ml of
The mutagenic activity data obtained in the Salmonella/microsome assay are shown in Table 1. The table shows the presence of mutagenicity induction for the aqueous extract in the presence of S9 and signs of mutagenicity in the absence of S9 for the TA102 strain, and signs of mutagenicity for TA100 in the presence of S9. The methanolic extract induced mutagenicity in TA102 (/S9 and /S9) and in TA100 (in the absence of S9, and signs of mutagenicity in the presence of S9). Higher mutagenic indexes (namely, ratio between the average of the revertant number induced per plate for each concentration and the average of the number of revertants of the negative control) were obtained in the presence of metabolic activation in TA102, which indicate the presence of oxidative mutagens (Table 2). Table 1 also shows that the methanolic extract at the concentration of 13.5 mg/plate presented signs of toxicity for TA100 and TA97a. Concentrations above 20.25 mg/plate were toxic for all strains because, the revertant frequencies were below the normal spontaneous frequency. Mutagenicity was not observed in any strain when the n-butanolic fraction was evaluated in the presence or absence of S9. 3.2. Chromosomal aberrations The results obtained through analysis of chromosomal aberrations and mitotic index for CHO cells treated
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260
Table 1 Mutagenic activity of aqueous and methanolic extracts and of the n -butanolic fraction from B. gaudichaudii on S. typhimurium strains Extract/fraction
Dose mg/plate
Revertants his/plate (mean9S.D.)a TA102
TA100
TA98
TA97a
S9
S9
S9
S9
S9
S9
S9
S9
Aqueous extract
3.37 6.75 10.12 13.50 20.25 27.00
255962 369953 313933 388946* 407914** 336956
521997 540935** 644920** 7289102** 10209100** 9659378
188921 178933 / 196913 191905 199927
227938 213908* 215907* 273963 331966* 298957*
30907 34906 / 39906 39909 45910
55908 61904 61901 66917 54921 41904
159928 151930 / 156935 165941 155933
/ 228922 / 292926 283961 210962
Methanolic extract
3.37 6.75 10.12 13.50 20.25
6019214 418975 9309339* 5069158 /
1073990** 1353981** 7189275 8409180* /
430947** 417995* 341954* 64933 25924
281998 283913** 269915* 105911 /
49924 50936 47923 47933 2902
58916 40908 50932 36908 /
154914 187974 17916 / /
167962 133930 49927 10902 /
n -Butanolic fraction
3.37 6.75 10.12 13.50 20.25
249940 277913 287932 315933 228928
300922 314941 343919 353942 330925
175913 196905 176907 192909 149915
148903 130914 166924 154916 172916
27905 29904 29904 23903 19902
69903 54908 / 54905 52907
185907 179924 176920 171915 180914
191914 194914 198916 183920 217938
Negative
Control
276921
321922
179924
173906
36908
41905
157914
169928
Positive
Control
13219104
1015985
11179255
11049125
13289182
333939
1038982
11879110
Negative control 0.1 ml DMSO (extract solvent). Positive control 2-anthramine (0.625 mg/plate)0 TA100 and TA98 (S9); 2-aminofluorene (10 mg/plate)0 TA102 and TA97a (S9); daunomycin (3 mg/plate)0 TA102 (S9); sodium azide (1.25 mg/plate)0 TA100 (S9); 4-nitro-o phenylenediamine (5 mg/plate)0 TA98 and TA97a (S9). a Values represent the mean9S.D. of at least two experiments carried out in triplicate. * P B 0.05 (ANOVA). ** P B 0.01 (ANOVA). Table 2 Mutagenic indexes for the aqueous and methanolic extracts and for the n -butanolic fraction from B. gaudichaudii in S. typhimurium strains Treatment mg/plate
Mutagenic indexesa TA102
TA100
TA98
TA97a
S9
S9
S9
S9
S9
S9
S9
S9
Aqueous extracts 3.37 6.75 10.12 13.50 20.25 27.00
0.92 1.33 1.13 1.41 1.47 1.22
1.62 1.68 2.00 2.26 3.18 3.01
1.05 0.99 / 1.09 1.07 1.11
1.31 1.23 1.24 1.58 1.91 1.72
0.83 0.94 / 1.08 1.08 1.25
1.37 1.34 1.49 1.61 1.32 1.00
1.01 0.96 / 0.99 1.05 0.92
/ 1.35 / 1.24 1.67 1.24
Methanolic extract 3.37 6.75 10.12 13.50 20.25
2.18 1.51 3.37 1.83 /
3.34 4.21 2.24 2.62 /
2.40 2.33 1.91 0.36 0.14
1.62 1.64 1.55 0.61 /
1.36 1.39 1.31 1.31 0.05
1.41 0.98 1.22 0.88 /
0.98 1.19 0.11 / /
0.99 0.79 0.29 0.06 /
n-Butanolic fraction 3.37 6.75 10.12 13.50 20.25
0.90 1.00 1.03 1.14 0.82
1.07 0.98 1.07 1.10 1.03
0.98 1.09 0.98 1.07 0.83
0.86 0.75 0.96 0.89 0.99
0.75 0.80 0.80 0.64 0.53
1.68 1.32 / 1.32 1.27
1.18 1.14 1.12 1.09 1.15
1.13 1.15 1.17 1.08 1.28
a
Mutagenic index (MUI): number of his (revertant colonies) induced in the sample/number of spontaneous his in the negative control. Mutagenic index in bold type indicates that the number of revertants is twice of the negative controls yields.
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261
Table 3 Distribution of the different types of chromosomal aberrations and mitotic index observed in CHO cells after treatment with methanolic extract of B. gaudichaudii in different times (12, 8 and 3 h) during the cell cycle Treatment (mg/ml)
Chromosomal aberrations Gap
Break
Total
NCA
MI (%)
OTA
c
ic
c
ic
12 h (G1 /S) Control DMSO 0.4 0.8 1.2
21 18 62 46 63
5 5 13 5 7
7 3 3 7 39
/ / / 1 12
1d 1t, 1d 2t, 3d 1t 14t, 1d
34 28 83* 60* 136*
32 36 67* 53* 92*
4.50 4.00 5.10 4.40 2.00*
8 h (S phase) Control DMSO 0.4 0.8 1.2
13 21 12 21 24
4 3 4 3 10
6 2 20 45 40
3 1 5 5 7
/ 1t, 1d / 7t 16t, 1d
26 29 41* 81* 98*
24 28 34* 73* 98*
6.00 5.00 5.30 6.50 4.40
3 h (G2 /S) Control DMSO 0.4 0.8 1.2
9 24 19 9 27
/ / 1 12 /
12 6 9 39 34
/ 1 4 5 1
/ 2f, 3t 2t 3t, 1q 5t, 2f, 2q
21 36 35 69 71
21 31 32 60 60
6.70 7.80 5.60* 4.30* 2.10*
Three hundred cells were analyzed per treatment; c, chromatid-type; ic, isochromatid-type; d, dicentric; f, fragments; q, quadriradial; t, triradial; NCA, number of cells with aberrations; MI, mitotic index; OTA, other types of aberrations. * Statistically different when compared with solvent control.
Table 4 Distribution of the different types of chromosomal aberrations and mitotic index observed in CHO cells after treatment with aqueous extract of B. gaudichaudii in different times (12, 8 and 3 h) during the cell cycle Treatment (mg/l)
Chromosomal aberrations Gap
Break c
Total
NCA
MI (%)
OTA
c
ic
ic
12 h (G1 /S) Control DMSO 1.2 1.4 1.6
15 16 29 26 16
3 2 3 6 3
6 8 5 2 /
1 / 5 1 1
/ 1t 1t 1q 3t, 1r, 1d
25 27 43 36 25
25 26 35 35 22
5.33 5.53 4.87 4.10 5.53
8 h (S) Control DMSO 1.2 1.4 1.6
7 6 16 29 18
5 1 3 1 6
16 2 11 4 15
3 / 3 4 4
1d / 3t, 1q 2r, 2d, 3t 2d, 2t
32 9 37 45 47
29 9 35 37 44
6.63 5.87 5.80 4.40 6.00
3 h (G2 /S) Control DMSO 1.2 1.4 1.6
8 11 9 12 28
1 3 2 / 4
5 16 11 6 11
/ 1 1 / 1
1q / 1t / 2d, 4t, 1q
15 31 24 18 56
15 29 22 17 54
6.00 6.30 5.03 4.40 3.23
Three hundred cells were analyzed per treatment; c, chromatid-type; ic, isochromatid-type; d, dicentric; q, quadriradial; t, triradial; r, ring; NCA, number of cells with aberrations; MI, mitotic index; OTA, other types of aberrations.
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262
in different times during the cell cycle are shown in Tables 3 and 4. Treatment with the various concentrations (1.2 /1.6 mg/ml) of the aqueous extract of Brosimum (Table 4) caused no significant increase in the number of chromosomal aberrations, in the frequency of cells with aberrations or in mitotic index. In contrast, treatment with the methanolic extract (Table 3) at all concentrations used (0.4 /1.2 mg/ml) caused a significant increase (P B/ 0.05) in the number of cells with chromosomal aberrations and in the frequency of such cells when they were treated in the G1/S and S phases. The mitotic index decreased significantly with increasing methanolic extract concentrations in the G2/S phase, and in the G1/S phase when the highest concentration was used. The most frequent aberrations were chromatid gaps and breaks. Other types of aberrations such as isochromatid gaps and breaks, quadriradial and triradial figures, dicentrics and fragments were also observed. When statistical analysis was conducted without considering gaps, the results obtained were the same.
4. Discussion and conclusions The methanolic extract of the plant induced a significant increase in the number of revertants for the TA100 strain in the absence of S9, and signs of mutagenicity in the presence of S9, and for TA102, in the presence and absence of S9. The aqueous extract only induced a significant increase in number of revertants in TA102 and signs of mutagenicity in TA100 in the presence of S9. The n-butanolic fraction did not induce mutagenicity. The results clearly showed an increase in chromosomal aberrations in the cultures treated with the methanolic extract in the G1/S and S phases of the cell cycle. In the G2/S phase an increase also occurred in the number of aberrations and in the frequency of cells with aberrations, which, however, was not significant. The mitotic index was significantly reduced in cultures treated in the G1/S phase with 1.2 mg/ml concentration, and in cultures treated in the G2/S phase with all
concentrations tested. Table 5 summarizes the results on the genotoxicity of B. gaudichaudii . The psoralens are tricyclic compounds consisting of a furan ring fused with a coumarin. According to Lagatolla et al. (1998), they cause countless biological effects, but the more commonly accepted one is the effect on DNA. The psoralens are secondary plant metabolites found in many fruits and vegetables. Synthetic forms of 5methoxypsoralen (bergapten) and 8-methoxypsoralen (8-MOP) have been used in combination with UV radiation in skin photochemotherapy for decades. However, handling or ingestion of psoralen-containing plants as well as medicinal use of these compounds has been shown to cause human health hazards (dermatitis, hepatotoxicity) (Diawara et al., 2000). The reaction of 8-MOP with DNA starts by a process of intercalation of the chemical compound between the stacked bases of DNA. In the absence of light, no covalent binding is established between the two molecules. However, absorption of near-UV light (320 /400 nm) allows 8-MOP to react either through its furan or pyrone moiety with a pyrimidine (preferably thymine) in one DNA strand, yielding monoadducts. Further absorption of light in the same wavelength range allows the reaction of some of the monoadducts */those already formed by the furan side of the psoralen*/ with another pyrimidine placed in an adjacent position on the opposite DNA strand, thus, yielding interstrand cross-links (BenHur and Song, 1984; Cimino et al., 1985) Psoralens are agents that intercalate in the DNA molecule forming mono- and di-adducts, and they also induce the production of singlet oxygen and superoxide radicals (Pathak and Joshy, 1984; Bianchi et al., 1996) that are oxidative mutagens clearly detected by the TA102 strain. This strain has an A /T base-pair at the critical site for reversion; it detects a variety of oxidative mutagens, active forms of oxygen, and has an intact excision-repair pathway (Levin et al., 1982). Furocoumarins appear to be able to generate 1O2 even when complexed with DNA (DeMol et al., 1981). The
Table 5 Summary of results on the genotoxicity of B. gaudichaudii Extract/fraction
Salmonella assaysa TA102
Aqueous extract Methanolic extract n -Butanolic fraction
CHO assays
TA100
TA98
TA97a
CA
MI
S9
S9
S9
S9
S9
S9
S9
S9
G1/S
S
G2/S
G1/S
S
G2/S
9
9 9
nt
nt
nt
nt
nt
nt
, Positive (ANOVA, P 0 0.05; MUIE 2); , negative; 9, signs of positive response (ANOVA, P B 0.05; MUIB 2); nt, not tested; CA, chromosomal aberrations; MI, mitotic index; G1/S, S, G2/S, cell cycles phases. a S9, without metabolic activation; S9, with metabolic activation.
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mutagenic effect of 1O2 may even be enhanced in the immediate proximity of DNA because of its longer life time in the hydrophobic DNA regions (Merkel and Kearns, 1972; Ito, 1980). Furthermore, 1O2 formed outside DNA by free furocoumarins in the cytoplasm or by furocoumarins noncovalently bound to proteins may contribute to the genetic effects, because diffusion of 1O2 over relatively large portions of the cells seems possible (Pooler and Valenzeno, 1979; Ito, 1980). Gamper et al. (1987) and Boyer et al. (1988) showed that psoralens react preferentially with thymidine residues, with interstrand cross-link formation strongly favored at 5?-TpA-3? sites. A 5?-TpA-3? site exists adjacent to the hisG46 codon (Hartman et al., 1986) of S. typhimurium strain TA100. According to Levin and Ames (1986) and Koch (1986), psoralen induced base-pair substitution in Salmonella . In addition, we observed mutagenic activity in the TA100 Salmonella strain which detects mutagens that cause base-pair substitutions (Maron and Ames, 1983). Considering that furocoumarins, bergapten and psoralen, are the major components (60%) of the methanolic extract of B. gaudichaudii , it can be inferred that both the mutagenicity and clastogenicity observed might have been induced by these agents. The result obtained with the aqueous extract was less marked, probably because in the process of aqueous extraction the amount of furocoumarins (3%) removed from the plant was much smaller than that obtained by methanol extraction. This was probably the reason why the increase in the chromosomal aberrations in CHO cells exposed to the aqueous extract was not significant and was also less marked in the assays with Salmonella . HPLC analysis (data not shown) revealed that the n butanol fraction contained only traces of furocoumarins. This was expected, since furocoumarins were previously removed by the partition with chloroform. The absence of mutagenicity in the n -butanol fraction thus reinforces the hypothesis that furocoumarins are the components responsible for the mutagenicity. In fact, the literature reports a remarkable mutagenicity (Bianchi et al., 1996; Cebula and Koch, 1990) as well as toxicity (Ivie, 1987) of furocoumarins. Even though the culture dishes and bottles for the bacteria were incubated in the dark, all the experiments were performed in the presence of natural light, a fact that might have allowed the photoactivation of the furocoumarins. As observed in the present study, extracts of the root bark of B. gaudichaudii showed mutagenic activity in the S. typhimurium test and on CHO cells. Thus, caution should be taken in using these roots for the treatment of vitiligo in popular medicine. Additional studies are needed to identify other possible substances that might contribute to the effects observed.
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Acknowledgements The authors greatly thank FAPESP, CNPq and PADC-FCF-UNESP for their financial support and Prof. A.T. Natarajan (University of Leiden, The Netherlands) that supplied CHO cells (CHO9 line).
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