Mutation Research, 232 (1990) 37-43 Elsevier
37
MUT 02107
Genotoxic, mutagenic and recombinogenic effects of rauwolfia alkaloids G i l s a n e v o n P o s e r 1, H e l o i s a H . R . A n d r a d e 2, K a t i a V . C . L . d a Silva 3, A m r l i a T. H e n r i q u e s 1 a n d J 0 5 0 A.P. H e n r i q u e s 3 1 Curso de Pfs-Gradua~o em Farmdtcia e Departamento de Produ~o de Mat~ria-Prima da Faculdade de Farmdtcia, UFRGS, 90410 Porto Alegre, RS (Brazil), 2 Departamento de Gen~tica and 3 Departamento Fisiologia, Farmacologia e Biofisica, lnstituto de Biocigncias, UFRGS, 90049 Porto Alegre, R S (Brazil) (Accepted 25 February 1990)
Keywords: Reserpine; Ajmalicine; SOS Chromotest; Yeast
Summary In the last decade, the possible correlation between the use of reserpine and rauwolfia drugs as anti.hypertensive agents and breast cancer incidence has been investigated. For the purpose of evaluating the mutagenic and genotoxic effects of these drugs, reserpine and ajmalicine were studied using the SOS Chromotest and the induction of gene conversion, crossing-over and reverse mutation in the yeast diploid strain XS2316. The results indicated a lack of genotoxic, mutagenic and recombinogenic effects.
Reserpine, a rauwolfia alkaloid, has been used for many decades as an efficient antihypertensive agent (Wilkins et al., 1949). However, 3 case-control studies reported a possible association between the use of reserpine and rauwolfia derivatives and the occurrence of breast cancer (Boston Collaborative Drug Surveillance Program, 1974). A number of other studies, however, do not support this association (International Agency for Research on Cancer, 1980; Labarthe and O'FalIon, 1980; Curb et al., 1982; Friedman, 1983; Stanford et al., 1986). WHO still maintains this alkaloid and its derivatives in the Model List of Essential Drugs (World Health Organization, 1983). Taking into account that even the smallest risk should be considered and that the evidence came
Correspondence: Jo~o Antonio PSgas Hem-iques, Bio 3 - Setor de Biofisica, lnstituto de Bioei~neias - UFRGS, Rua Sarmento Leite, 500, 90049 - Porto Alegre, RS (Brazil).
from case-control studies, it is important to analyze these substances for their genotoxic properties. It has been suggested that alkaloids can be considered potentially mutagenic, since the therapeutic action of certain alkaloids is related to their interaction with DNA (Beljanski and Beljanski, 1982; Moustacchi et al., 1983; Melo et al., 1986) and the mutagenic and carcinogenic effects are due to this interaction (Miller and Miller, 1981). Therefore, it is important to ascertain if reserpine or rauwolfia derivatives exhibit genotoxic and mutagenic activities in prokaryotic and eukaryotic cells. This is important because in long-term risk estimates the correlation between carcinogenic and mutagenic activities is well documented (McCann et al., 1975). From these observations, it seemed very interesting to analyze the possible genotoxic effects of the alkaloids reserpine and ajmalicine, usually found in the Rauwolfia genus, by means of the SOS Chromotest described by Qnillardet et al. (1982). Several studies have indicated that most of the
0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
38 chemical mutagens are potential inducers of mitotic recombination (Zimmermann, 1971; Fahrig, 1973, 1976, 1979; Davies et al., 1975; Murthy, 1979; Kunz et al., 1980). In somatic tissues, it has been proposed that mitotic recombination, which can lead to homozygosity of recessive alleles, could be involved in the promotional stage of carcinogenesis (KinseUa and Radman, 1978) or in a cocarcinogenic process (Kunz et al., 1980). Thus, the diploid strain of Saccharomyces cerevisiae XS2316 (Machida and Nakai, 1980), which allows the simultaneous detection of the induction of mutation and the 2 forms of mitotic recombination (crossing-over and gene conversion), was employed to determine the possible mutagenic and recombinogenic activities of these alkaloids in a eukaryotic system.
MeO
I H
N
H
..H
OMe 0 -- ~10~OMe
OMe
OMe (z)
.Me
MeOOC~
Materials and methods (TT)
Strains Escherichia coli PQ37, which exhibits the following genotype F-, thr leu his-4 pyrD thi galE galk lac 169 srl300::Tn 10 rpoB rpsL uvrA rfa trp: : M UC sulA: : Mud (A P, lac ) c-ts, was used for the SOS Chromotest (Quillardet et al., 1982). This strain is constitutive for alkaline phosphatase synthesis. The diploid S. cerevisiae strain XS2316 has been previously described (Machida and Nakai, 1980) and its genotype is as follows: + /eul-1 trp5-48 + + + a hisl-1 ade6 1eu1-12 + cyh2 metl3 lys5-1 his1-1
Media The complete liquid medium (YEPD) contained 0.5% yeast extract (Difco, U.S.A.), 2% bactopeptone (Difco) and 2% glucose. The minimal medium (MM) contained 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose and 2% bacto-agar (Difco). The synthetic complete medium (SC) was MM supplemented with 2 mg adenine, 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine and 2 mg tryptophan, per 100 ml MM. The omission media ( S C - leucine, S C histidine, SC - methionine and SC - adenine) were a series of media in which one of the amino
Fig. 1. Chemical structure of the alkaloids reserpine (I) and ajmalicine(II).
acids or bases had been omitted from SC, e.g., SC minus leucine etc. The cycloheximide medium (SC + cyh) was SC supplemented with 200 /~g cycloheximide (Calbiochem, Los Angeles, CA, U.S.A.) per 100 ml SC. The LB medium contained 1% bactopeptone (Difco), 0.5% yeast extract (Difco) and 1% NaC1. Chemical products The alkaloids reserpine and ajmalicine (Fig. 1) were obtained from Merck (Darmstadt, F.R.G.). Their purity levels were chromatographically tested. To carry out SOS Chromotests, the alkaloids were diluted in spectrophotometric-grade dimetyl sulfoxide (DMSO). For the treatment of yeast cells, 1-mg/ml stock solutions of reserpine (MW.608.HC1) and of ajmalicine (MW.352.HC1) hydrochlorides, to which was added 0.2 ml of absolute ethanol, were prepared immediately prior to use. The appropriate solvent control included in the genetic tests was found to be negative. SOS Chromotest The SOS Chromotest was performed according to Quillardet et al. (1982). An exponential-phase
39 culture of PQ37, grown in LB medium plus ampicillin (20/~g/mi) at 37 o C, was diluted 1 : 10 into fresh medium or in a mixture of $9 mix for metabolic activation. Fractions (0.6 mi) were distributed into glass test tubes containing 20 /tl of the alkaloids to be tested. After 2-h incubation at 37 °C with shaking, a 0.3-ml sample was used to assay fl-galactosidase and alkaline phosphatase activities. The SOS induction factor in treated cells was obtained from the rate of fl-galactosidase and alkaline phosphatase activities compared to untreated cells. As positive controls, 4-nitroquinoline-l-oxide (4NQO) and aflatoxin B1) (AFB1) (both from Sigma Chemical, St. Louis, MO, U.S.A.) were used for the tests without and with metabolic activation, respectively. Microsomal fraction The $9 fraction was prepared from livers of Sprague-Dawley rats pretreated with 3-methylcholanthrene, purchased from Bionetics (Litton, Charleston, IL, U.S.A.). The $9 mix metabolic activation mixture was prepared according to Maron and Ames (1983). Yeast growth conditions Stationary-phase cultures were obtained by inoculation of 5 × 106 cells of a liquid YEPD culture in stationary phase into 5 ml of YEPD medium. After 48-h incubation at 30 °C with aeration by shaking, the cultures contained 4 × 108 cells/ml. Exponential-phase cultures were obtained by inoculation of 5 × 105 cells of the same YEPD culture in stationary phase into 5 ml of YEPD medium. After 12-h incubation in the same conditions, the cultures contained 2 × 107 cells/ml. The stationary- and exponential-phase cells were harvested, washed 3 times with phosphate buffer (0.067 M, pH 7), and were sonicated for 15 s in a 100-W ultrasonic disintegrator (INPEC, Sao Paulo, Brazil). Both the cell concentration and the percentage of budding cells in each culture were checked by microscopic counting. Test for the induction of mitotic recombination and mutational events following treatment with reserpine and ajmalicine A suspension of yeast cells in stationary (4 × 108
cells/ml) and exponential growth phase (4 × 107 cells/ml) were incubated for 20 h at 30 °C with various concentrations of reserpine and ajmalicine. The reaction was stopped by adding an equal volume of 4% thiosulfate. After treatment, the cells were diluted in phosphate buffer (0.067 M, pH 7.0), plated on 4 kinds of medium (SC, SC - leu, SC + cyh and SC - his) and incubated for 7-8 days at 30°C. This was followed by the examination of cell killing on SC, intragenic recombination (gene conversion on SC - leu, intergenic recombination (crossing-over) on SC + cyh and reverse mutation on S C - his. In order to measure the exact frequency of reciprocal crossing-over, it is necessary to eliminate the possibility that some cycloheximide-resistant colonies had resulted from reversion at the CYH2 locus, as well as monosomy on chromosome VII. For this purpose, the cycloheximide-resistant colonies were replica-plated on a series of media, S C - lys, SC - m e t and S C - ade, for screening the linked markers of CYH2. For each agent used, experiments were repeated at least 3 times. Platings were repeated at least in triplicate for each dose so that a minimum of 200 survivors and 50 recombinants per point was scored. For analysis of reverse mutation, platings were done 6 times for each dose. Treatment with 254-nm UV radiation The suspension of cells (4 × 108/ml) was irradiated with different doses of 254-nm UV radiation, as previously described by Henriques and Moustacchi (1980). Results and discussion
Table 1 shows that, in contrast to data obtained for the positive control 4NQO, the alkaloids reserpine and ajmalicine dearly did not induce flgalactosidase synthesis in the SOS Chromotest without metabolic activation. However, higher amounts (5 × 103 ng/test) of reserpine inhibited the constitutive enzyme alkaline phosphatase, which indicates a toxic effect of the treatment. Thus, the observed increase in the enzymatic ratio may be due to the loss of alkaline phosphatase activity and not to true SOS induction. In the presence of metabolic activation mixture, these 2 alkaloids were not genotoxic (Table 2).
40 TABLE 1 RESPONSE TO R E S E R P I N E A N D A J M A L I C I N E IN T H E SOS C H R O M O T E S T W I T H O U T M E T A B O L I C A C T I V A T I O N Agent
Dose (ng/test)
/3-Galactosidase (B) (units)
Alkaline phosphatase (P) (units)
B P
Induction factor b
4NQO a
0 4 10 100
174 273 658 2 430
1625 1 705 1 765 1687
0.107 0.160 0.373 1.440
1 1.50 3.48 13.46
Reserpine
0 1 × 102 1 ×102 2.5 × 103 4 :,
37
MUT 02107
Genotoxic, mutagenic and recombinogenic effects of rauwolfia alkaloids G i l s a n e v o n P o s e r 1, H e l o i s a H . R . A n d r a d e 2, K a t i a V . C . L . d a Silva 3, A m r l i a T. H e n r i q u e s 1 a n d J 0 5 0 A.P. H e n r i q u e s 3 1 Curso de Pfs-Gradua~o em Farmdtcia e Departamento de Produ~o de Mat~ria-Prima da Faculdade de Farmdtcia, UFRGS, 90410 Porto Alegre, RS (Brazil), 2 Departamento de Gen~tica and 3 Departamento Fisiologia, Farmacologia e Biofisica, lnstituto de Biocigncias, UFRGS, 90049 Porto Alegre, R S (Brazil) (Accepted 25 February 1990)
Keywords: Reserpine; Ajmalicine; SOS Chromotest; Yeast
Summary In the last decade, the possible correlation between the use of reserpine and rauwolfia drugs as anti.hypertensive agents and breast cancer incidence has been investigated. For the purpose of evaluating the mutagenic and genotoxic effects of these drugs, reserpine and ajmalicine were studied using the SOS Chromotest and the induction of gene conversion, crossing-over and reverse mutation in the yeast diploid strain XS2316. The results indicated a lack of genotoxic, mutagenic and recombinogenic effects.
Reserpine, a rauwolfia alkaloid, has been used for many decades as an efficient antihypertensive agent (Wilkins et al., 1949). However, 3 case-control studies reported a possible association between the use of reserpine and rauwolfia derivatives and the occurrence of breast cancer (Boston Collaborative Drug Surveillance Program, 1974). A number of other studies, however, do not support this association (International Agency for Research on Cancer, 1980; Labarthe and O'FalIon, 1980; Curb et al., 1982; Friedman, 1983; Stanford et al., 1986). WHO still maintains this alkaloid and its derivatives in the Model List of Essential Drugs (World Health Organization, 1983). Taking into account that even the smallest risk should be considered and that the evidence came
Correspondence: Jo~o Antonio PSgas Hem-iques, Bio 3 - Setor de Biofisica, lnstituto de Bioei~neias - UFRGS, Rua Sarmento Leite, 500, 90049 - Porto Alegre, RS (Brazil).
from case-control studies, it is important to analyze these substances for their genotoxic properties. It has been suggested that alkaloids can be considered potentially mutagenic, since the therapeutic action of certain alkaloids is related to their interaction with DNA (Beljanski and Beljanski, 1982; Moustacchi et al., 1983; Melo et al., 1986) and the mutagenic and carcinogenic effects are due to this interaction (Miller and Miller, 1981). Therefore, it is important to ascertain if reserpine or rauwolfia derivatives exhibit genotoxic and mutagenic activities in prokaryotic and eukaryotic cells. This is important because in long-term risk estimates the correlation between carcinogenic and mutagenic activities is well documented (McCann et al., 1975). From these observations, it seemed very interesting to analyze the possible genotoxic effects of the alkaloids reserpine and ajmalicine, usually found in the Rauwolfia genus, by means of the SOS Chromotest described by Qnillardet et al. (1982). Several studies have indicated that most of the
0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
38 chemical mutagens are potential inducers of mitotic recombination (Zimmermann, 1971; Fahrig, 1973, 1976, 1979; Davies et al., 1975; Murthy, 1979; Kunz et al., 1980). In somatic tissues, it has been proposed that mitotic recombination, which can lead to homozygosity of recessive alleles, could be involved in the promotional stage of carcinogenesis (KinseUa and Radman, 1978) or in a cocarcinogenic process (Kunz et al., 1980). Thus, the diploid strain of Saccharomyces cerevisiae XS2316 (Machida and Nakai, 1980), which allows the simultaneous detection of the induction of mutation and the 2 forms of mitotic recombination (crossing-over and gene conversion), was employed to determine the possible mutagenic and recombinogenic activities of these alkaloids in a eukaryotic system.
MeO
I H
N
H
..H
OMe 0 -- ~10~OMe
OMe
OMe (z)
.Me
MeOOC~
Materials and methods (TT)
Strains Escherichia coli PQ37, which exhibits the following genotype F-, thr leu his-4 pyrD thi galE galk lac 169 srl300::Tn 10 rpoB rpsL uvrA rfa trp: : M UC sulA: : Mud (A P, lac ) c-ts, was used for the SOS Chromotest (Quillardet et al., 1982). This strain is constitutive for alkaline phosphatase synthesis. The diploid S. cerevisiae strain XS2316 has been previously described (Machida and Nakai, 1980) and its genotype is as follows: + /eul-1 trp5-48 + + + a hisl-1 ade6 1eu1-12 + cyh2 metl3 lys5-1 his1-1
Media The complete liquid medium (YEPD) contained 0.5% yeast extract (Difco, U.S.A.), 2% bactopeptone (Difco) and 2% glucose. The minimal medium (MM) contained 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose and 2% bacto-agar (Difco). The synthetic complete medium (SC) was MM supplemented with 2 mg adenine, 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine and 2 mg tryptophan, per 100 ml MM. The omission media ( S C - leucine, S C histidine, SC - methionine and SC - adenine) were a series of media in which one of the amino
Fig. 1. Chemical structure of the alkaloids reserpine (I) and ajmalicine(II).
acids or bases had been omitted from SC, e.g., SC minus leucine etc. The cycloheximide medium (SC + cyh) was SC supplemented with 200 /~g cycloheximide (Calbiochem, Los Angeles, CA, U.S.A.) per 100 ml SC. The LB medium contained 1% bactopeptone (Difco), 0.5% yeast extract (Difco) and 1% NaC1. Chemical products The alkaloids reserpine and ajmalicine (Fig. 1) were obtained from Merck (Darmstadt, F.R.G.). Their purity levels were chromatographically tested. To carry out SOS Chromotests, the alkaloids were diluted in spectrophotometric-grade dimetyl sulfoxide (DMSO). For the treatment of yeast cells, 1-mg/ml stock solutions of reserpine (MW.608.HC1) and of ajmalicine (MW.352.HC1) hydrochlorides, to which was added 0.2 ml of absolute ethanol, were prepared immediately prior to use. The appropriate solvent control included in the genetic tests was found to be negative. SOS Chromotest The SOS Chromotest was performed according to Quillardet et al. (1982). An exponential-phase
39 culture of PQ37, grown in LB medium plus ampicillin (20/~g/mi) at 37 o C, was diluted 1 : 10 into fresh medium or in a mixture of $9 mix for metabolic activation. Fractions (0.6 mi) were distributed into glass test tubes containing 20 /tl of the alkaloids to be tested. After 2-h incubation at 37 °C with shaking, a 0.3-ml sample was used to assay fl-galactosidase and alkaline phosphatase activities. The SOS induction factor in treated cells was obtained from the rate of fl-galactosidase and alkaline phosphatase activities compared to untreated cells. As positive controls, 4-nitroquinoline-l-oxide (4NQO) and aflatoxin B1) (AFB1) (both from Sigma Chemical, St. Louis, MO, U.S.A.) were used for the tests without and with metabolic activation, respectively. Microsomal fraction The $9 fraction was prepared from livers of Sprague-Dawley rats pretreated with 3-methylcholanthrene, purchased from Bionetics (Litton, Charleston, IL, U.S.A.). The $9 mix metabolic activation mixture was prepared according to Maron and Ames (1983). Yeast growth conditions Stationary-phase cultures were obtained by inoculation of 5 × 106 cells of a liquid YEPD culture in stationary phase into 5 ml of YEPD medium. After 48-h incubation at 30 °C with aeration by shaking, the cultures contained 4 × 108 cells/ml. Exponential-phase cultures were obtained by inoculation of 5 × 105 cells of the same YEPD culture in stationary phase into 5 ml of YEPD medium. After 12-h incubation in the same conditions, the cultures contained 2 × 107 cells/ml. The stationary- and exponential-phase cells were harvested, washed 3 times with phosphate buffer (0.067 M, pH 7), and were sonicated for 15 s in a 100-W ultrasonic disintegrator (INPEC, Sao Paulo, Brazil). Both the cell concentration and the percentage of budding cells in each culture were checked by microscopic counting. Test for the induction of mitotic recombination and mutational events following treatment with reserpine and ajmalicine A suspension of yeast cells in stationary (4 × 108
cells/ml) and exponential growth phase (4 × 107 cells/ml) were incubated for 20 h at 30 °C with various concentrations of reserpine and ajmalicine. The reaction was stopped by adding an equal volume of 4% thiosulfate. After treatment, the cells were diluted in phosphate buffer (0.067 M, pH 7.0), plated on 4 kinds of medium (SC, SC - leu, SC + cyh and SC - his) and incubated for 7-8 days at 30°C. This was followed by the examination of cell killing on SC, intragenic recombination (gene conversion on SC - leu, intergenic recombination (crossing-over) on SC + cyh and reverse mutation on S C - his. In order to measure the exact frequency of reciprocal crossing-over, it is necessary to eliminate the possibility that some cycloheximide-resistant colonies had resulted from reversion at the CYH2 locus, as well as monosomy on chromosome VII. For this purpose, the cycloheximide-resistant colonies were replica-plated on a series of media, S C - lys, SC - m e t and S C - ade, for screening the linked markers of CYH2. For each agent used, experiments were repeated at least 3 times. Platings were repeated at least in triplicate for each dose so that a minimum of 200 survivors and 50 recombinants per point was scored. For analysis of reverse mutation, platings were done 6 times for each dose. Treatment with 254-nm UV radiation The suspension of cells (4 × 108/ml) was irradiated with different doses of 254-nm UV radiation, as previously described by Henriques and Moustacchi (1980). Results and discussion
Table 1 shows that, in contrast to data obtained for the positive control 4NQO, the alkaloids reserpine and ajmalicine dearly did not induce flgalactosidase synthesis in the SOS Chromotest without metabolic activation. However, higher amounts (5 × 103 ng/test) of reserpine inhibited the constitutive enzyme alkaline phosphatase, which indicates a toxic effect of the treatment. Thus, the observed increase in the enzymatic ratio may be due to the loss of alkaline phosphatase activity and not to true SOS induction. In the presence of metabolic activation mixture, these 2 alkaloids were not genotoxic (Table 2).
40 TABLE 1 RESPONSE TO R E S E R P I N E A N D A J M A L I C I N E IN T H E SOS C H R O M O T E S T W I T H O U T M E T A B O L I C A C T I V A T I O N Agent
Dose (ng/test)
/3-Galactosidase (B) (units)
Alkaline phosphatase (P) (units)
B P
Induction factor b
4NQO a
0 4 10 100
174 273 658 2 430
1625 1 705 1 765 1687
0.107 0.160 0.373 1.440
1 1.50 3.48 13.46
Reserpine
0 1 × 102 1 ×102 2.5 × 103 4 :,
