Mutation Research, 260 (1991) 145-152 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100090U
145
MUTGEN 01652
Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms Paulo Roberto H. Moreno 1, Vera Maria F. Vargas 2, Heloisa Helena R. Andrade 3, Amelia T. Henriques 1 and Jogo Antonio P. Henriques 4 I Curso de Pds-Gradua~o em Farmdcia-UFRGS, 2 Departamento do Meio Ambiente, Secretaria da Saftde e do Meio Ambiente, Departamento de Fisiologia, Farmacologia e Biofisica and 4 Departamento de Gendtica, Instituto de Bioci~ncias-UFRGS, Porto Alegre, R S (Brazil) (Received 17 July 1990) (Revision received 2 November 1990) (Accepted 7 November 1990)
Keywords: Boldine; Ames test; SOS chromotest; Yeast
Summary The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.
Alkaloids are a large group of natural products recognized for their remarkable pharmacological activities. Among the different classes of alkaloids the aporphines have been pharmacologically studied with regard to their properties in the central nervous system (CNS) as dopamine agonists (Neumeyer, 1985). Aporphines have been used as 'rigid' analogues of dopamine in the determination of the interaction with the dopaminergic receptors (Neumeyer, 1985).
Correspondence: Dr. J.A. P~gas Henriques, Instituto de Bioci8ncias-UFRGS, Setor de Biofisica - BIO-3, Rua Sarmento Leite 500, 90049 Porto Alegre, RS (Brazil).
Some aporphine alkaloids could inhibit the growth of the microorganisms Scenedesmus obliquus (Jatimiliansky and Sivori, 1969). Boldine is an aporphine alkaloid usually found in the plant families Magnoliaceae, Annonaceae, Rhamnaceae and Monimiaceae (Guinaudeau et al., 1983, 1988). In the last one it has the species Peumus boldus (boldo-do-Chile), widely used in folk medicine all over the world (Duke, 1985). In human therapy preparations of medicinal plants are widely used although little information on their potential risk to health is available. It has been suggested that alkaloids can be considered potentially mutagenic, since the therapeutic action of certain alkaloids is related to its interaction with DNA (Beljansky and Beljansky, 1982;
146
Moustacchi et al., 1983; Melo et al., 1986; Von Poser et al., 1990) and the mutagenic and carcinogenic effects are due to this interaction (Miller and Miller, 1981). Therefore, it is yery important to ascertain if boldine, as a model of aporphine alkaloids, exhibits genotoxic and mutagenic activities in prokaryotic and eukaryotic cells. This is important for long-term risk estimates since 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 and mutagenic effects of boldine by means of the SOS chromotest (Quillardet and Hofnung, 1985) and the Ames test (Ames et al., 1975). Several studies have indicated that most chemical mutagens are potential inducers of mitotic recombination (Davies et al., 1975; Fahrig, 1979; Kunz et al., 1980; Von Poser et al., 1990). 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 Radmann, 1978) or in a cocarcinogenic process (Kunz et al., 1980). Thus, the diploid strain of Saccharomyces cereoisiae XS2316 (Machida and Nakai, 1980), which allows the detection of the 2 forms of mitotic recombination (crossing-over and gene conversion), was employed to determine the possible recombinogenic activities of this alkaloid in a eukaryotic system. The boldine alkaloid was also tested for induction of 2 types of locus-specific mutation in haploid yeast cells, reversion of the lysl-1 ochre and his4-38 frameshift alleles. Moreover, boldine was tested for its ability to induce mitochondrial 'petite' mutations. Materials and methods
Strains Escherichia coli SOS-chromotest tester strain PQ37 (as described by QuiUardet and Hofnung, 1985) was obtained from Dr. P. Qnillardet. The Salmonella typhimurium strains TA98, TA100 and TA102, as described by Maron and Ames (1983), were kindly provided by Dr. B.N. Ames. The haploid strain of Saccharomyces cereoisiae BM7126-9A (a leul-I ade2-1 his4-38 leu2-3 ura rho °) was constructed by Bankmann and Brendel
4
CH30
~
NCH 3 "H
(
c%o,
OH
BOLDINE
Fig. 1. Chemical structure of the aporphine alkaloid boldine.
(1989). The N123 (a his1 ) haploid yeast strain was kindly provided by Dr. E. Moustacchi. The diploid S. cereuisiae strain XS2316 has the following genotype (Machida and Nakai, 1980): + leul-1 trp5-48 + + + a his1-1 ade6 ° leul-12 + cyh2 m e t l 3 lys5-1 a his1-1
Chemical products The aporphine alkaloid boldine (Fig. 1) was obtained from Sigma Co. (St. Louis, MO, U.S.A.). Its purity level was confirmed by 2 different chromatographic systems. To carry out the SOS chromotest and the Ames test the alkaloid was diluted in spectrophotometric-grade dimethyl sulfoxide (DMSO). For treatment of yeast cells a stock solution of 1 mg/mi boldine was prepared using the mixture saline solution (0.8 ml) and absolute ethanol (0.2 ml) immediately prior to use. The appropriate solvent controls included in the genetic tests were found to be negative. 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 methionine and SC - adenine) were a series of SC media in which one of the amino acids or bases had been omitted. The cycloheximide medium (SC + cyh) was SC supplemented with 200/xg cycloheximide (Calbiochem, U.S.A.) per 100 ml SC.
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For mutagenesis SC medium was MM supplemented with 3 mg each of lysine and leucine, 2 mg each of arginine, uracil, methionine, histidine, and tryptophan and 0.5 mg of adenine, per 100 ml MM. Omission media lacking lysine, histidine were sub-supplemented with 0.1 mg of lysine or histidine, respectively, per 100 ml omission medium. LB medium contained 1% bactopeptone (Difco), 0.5% yeast extract (Difco) and 1% NaC1. S O S chromotest The SOS chromotest was performed according to Quillardet et al. (1985). An exponential-phase culture of PQ37, grown in LB medium plus ampicillin (20 #g/ml) at 37°C, was diluted 1:10 into fresh medium or in mixture of $9 mix for metaboric activation. Fractions (0.6 ml) were distributed into glass test tubes containing 20 #1 of boldine. After 2-h incubation at 37 °C with shaking, 0.3-ml samples were used to assay /3-galactosidase and alkaline-phosphatase activities. SOS induction factor in treated cells was obtained from the ratio of /3-galactosidase and alkaline phosphatase activities, compared to untreated cells. As positive controls, 4-nitroquinoline 1-oxide (4NQO) and aflatoxin B1 (AFB1) (both from Sigma) were used for the tests without and with metabolic activation, respectively. Ames test Mutagenicity was assayed by the spot test procedure (Ames et al., 1975). Samples of boldine (0.5, 5, 50, 100 and 200/~g/plate) were spotted on a plate with 0.1 ml of tester bacterial cultures in the presence or absence of $9 mix (20 #1 of $9 fraction for 500/~1 of $9 mix) and incubated in the dark at 37°C for 48 li. For strain TA102 the mutagenicity was assayed by the preincubation procedure (Maron and Ames, 1983). Negative (DMSO) and positive (5 #g sodium azide per plate for TA100, 0.5 fig 4NQO per plate for TA98 and 100 /~g hydrogen peroxide for TA102) controls were included in each assay. 2-Aminoanthracene (2-AA, 0.1 txg per plate) for TA102 and AFB 1 (0.5 /~g per plate) for TA100 and TA98 were used as positive controls for the metabolic assays. Microsomal fraction The microsomal fraction $9 was prepared from livers of Sprague-Dawley rats pretreated with
polychlorinated biphenyl mixture (Aroclor 1254) as described by Maron and Ames (1983). The $9 mix metabolic activation mixture was prepared according to Quillardet and Hofnung (1985) for the SOS chromotest and according to Maron and Ames (1983) for the Ames test. Yeast growth conditions Cells were routinely cultured in YEPD from single-colony isolates. In each independent experiment, 2 X 106 cells/ml were seeded and shaken vigorously at 30 °C for 3 days to obtain cultures in the stationary phase of growth. Exponentialphase cultures were obtained by inoculation of 5 x 105 cells of the same stationary YEPD culture in 5 ml of YEPD medium. After 12 h of incubation in the same conditions, the cultures contained 2 x 107 cells/ml. Cells were harvested and washed twice with saline (0.9% NaC1). Clumps were dissociated by sonication of the cell suspension for 15 s in a Thornton ultrasonic disintegrator. The cell number and the percentage of budding cells were determined with a counting chamber. Detection of boldine-induced reverse mutation A suspension of cells (2 X 108 cells/ml) in exponential growth phase were incubated for 20 h at 30 ° C with various concentrations of boldine. After treatment, survival (on SC, 5-7 days, 30 ° C) and LYS or HIS prototrophic colonies (on appropriate omission media, 7-10 days, 30°C) were scored. HIS prototrophic revertants were picked onto SC - his medium and after 2 days at 30 ° C replicated onto S C - l e u medium. After 3 days at 30°C co-reversion to LEU prototrophy was scored. Both lysl-1 and ade2-1 are suppressible UAA (ochre) nonsense alleles (Eckardt and Haynes, 1977). Therefore, white-colored LYS-prototrophic mutants are, with high probability, locus-non-specific intergenic revertants (suppressors) whereas redcolored LYS-prototrophic colonies demonstrate locus-specific mutational events. Both his4-38 and leu2-3 alleles represent frameshift mutations (his438: + G C insertion) (Mathison and Culbertson, 1985) induced after treatment with ICR-170 (Culbertson et al., 1977) and are suppressible by group-II frameshift suppressors (Gaber and Culbertson, 1982). Thus, double prototrophic mutants induced in his4-38 leu2-3 strains can be referred
148 a n d i n c u b a t e d for 7 - 8 d a y s at 3 0 ° C . Colonies g r o w n o n S C m e d i u m y i e l d e d d a t a of cell survival a n d colonies g r o w n o n S C - leu a n d SC + cyh were scored for i n t r a g e n i c r e c o m b i n a t i o n (gene conversion) a n d i n t e r g e n i c r e c o m b i n a t i o n (crossing-over), respectively. I n o r d e r to m e a s u r e the exact f r e q u e n c y of r e c i p r o c a l crossing-over, it is necessary to e l i m i n a t e the p o s s i b i l i t y t h a t some c y c l o h e x i m i d e - r e s i s t a n t colonies h a d resulted f r o m reversion at the C Y H 2 locus, as well as m o n o s o m y on c h r o m o s o m e VII. F o r this p u r p o s e , the cycloh e x i m i d e - r e s i s t a n t colonies were r e p l i c a - p l a t e d on a series of m e d i a , S C - lys, S C - m e t a n d S C ade, for screening these l i n k e d m a r k e r s of cyh2. T h e e x p e r i m e n t was r e p e a t e d at least 3 times. Platings were d o n e in t r i p l i c a t e for each dose so t h a t a m i n i m u m o f 200 survivors a n d of 7 0 - 9 0 r e c o m b i n a n t s p e r p o i n t were scored.
to as suppressors whereas leu-auxotrophic H I S revertants ( a n d vice versa) r e p r e s e n t locus-specific revertants. T h e e x p e r i m e n t was r e p e a t e d at least 3 times.
Detection of cytoplasmic "petite' mutation T h e frequency of m i t o c h o n d r i a l r e s p i r a t o r y - d e ficient m u t a n t s or ' p e t i t e s ' was d e t e r m i n e d b y the t r i p h e n y l t e t r a z o l i u m overlay t e c h n i q u e ( O g u r et al., 1957).
Detection of induced mitotic recombination Suspensions of ceils in s t a t i o n a r y (2 × 10 8 c e l l s / m l ) a n d e x p o n e n t i a l p h a s e s (2 x 10 7 c e l l s / m l ) of g r o w t h were i n c u b a t e d for 20 h at 30 o C with various c o n c e n t r a t i o n s of b o l d i n e . A f t e r treatment, the cells were d i l u t e d in saline, p l a t e d o n 3 kinds of m e d i u m (SC, SC - leu a n d SC + cyh)
TABLE 1 SOS FUNCTION-INDUCING ACTIVITY OF BOLDINE IN Escherichia coli STRAIN PQ37 IN THE SOS CHROMOTEST Substance
Concentration (ng/test)
fl-Galactosidase(B) (units)
Alkaline phosphatase (P) (units)
B
Induction factor
Without metabolicactivation 4-NQO a
0 10 100
Boldine
0 5 1 5 1 5 1
×10 ~ x 102 x 102 x 103 x 103 x 104
109 347 1747
509 509 438
0.214 0.681 4.063
1 3.18 18.98
109 133 145 116 116 118 109
509 500 500 525 519 531 540
0.214 0.266 0.290 0.221 0.224 0.222 0.202
1 1.24 1.36 1.03 1.05 1.04 0.94
167 443 1018
1578 1629 1617
0.106 0.272 0.629
1 2.57 5.93
230 200 169 193 174 191 165
952 937 950 952 1020 990 984
0.230 0.213 0.178 0.203 0.171 0.193 0.168
1 0.93 0.77 0.88 0.74 0.84 0.73
With metabolicactivation AFB1 a
0 5 20
Boldine
a Positive controls.
0 5 x 102 1 x 103 2.5 x 10 3 5 × 103 7.5 x 103 1 × 104
149
8-Methoxypsoralen + UVA treatment 8-Methoxypsoralen (8-MOP) (Sigma) was used as positive control in the determination of mutational and recombinational events in yeast. The conditions were as previously described by Henriques et al. (1989). Results
Table 1 shows that boldine, in contrast to data obtained for positive controls, clearly did not induce SOS function (fl-galactosidase synthesis), when tested with the SOS chromotest with and without metabolic activation. These results suggest that this alkaloid does not have genotoxic activity. To search for mutagenicity of boldine we used the haploid yeast strain BM7126-9A which allows the simultaneous scoring of locus-specific mutation in 2 genes: point mutation in the ochre allele lysl-1 and frameshift mutation in the his4-38 allele; locus specificity of reversion is discriminated for by detection of intergenic suppression (co-reversion of ade2-1 for ochre suppression and of leu2-3 for group-II frameshift suppression). Table 2 shows that at the doses applied boldine was non-toxic and was not able to change significantly the frequency of reversion of both lysl-1 and
his4-38 alleles in comparison with the non-treated control. The same lack of mutagenic activity has also been found in the Ames test using strains TA100 (base-pair substitution), TA98 (frameshift mutation) and TA102 (detects oxidative and alkylating mutagens and active forms of oxygen; Lewin et al., 1982) with and without microsomal activation (data not shown). It can be also observed in Table 2 that the mitochondrial 'petite' mutation is significantly induced by the aporphine alkaloid boldine. The search for a recombinogenic effect of this alkaloid was carried out in stationary and exponential cultures of yeast diploid cells. Table 3 shows that at the doses applied the frequencies of convertants (LEU +) and recombinants (CYH +) induced by boldine were significantly increased (Student's t-test). The induction of gene conversion in the exponentially growing diploid yeast cells was higher than crossing-over. In fact it corresponds to a 5-6-fold increase of convertogenic events over the spontaneous background. Although the frequency of both convertants and recombinants in stationary diploid cells was less pronounced to that observed in exponential-phase cells, both induction kinetics were practically the same (data not shown).
TABLE 2 I N D U C T I O N OF F R A M E S H I F T (his4-38), P O I N T M U T A T I O N (lysl-1, O C H R E A L L E L E ) A N D C Y T O P L A S M I C 'PETITE' M U T A T I O N S IN H A P L O I D YEAST STRAINS, A F T E R B O L D I N E T R E A T M E N T Agent
8-MOP a + UVA Boldine
Dose
Concentration
Survival (Z)
(kJ//m2)
(ttg/ml)
N123
0 2.0
-
-
0 25 50 100 125 150 200
100 94 75 74 77
(933) (879) (696) (693) (735)
his4/lO 7 survivors d
l y s l / l O 7 survivors d
'Petite'
BM7126-9A
BM7126-9A
BM7126-9A
m u t a n t s (%) N123
100(672) 18 (273)
0.29+0.22 b (2) c 1.0 +0.37 (27)
1.80_+ 0.72 (16) 115.13_+10.61 (188)
100 101 90 85 87 80 77
0.26 0.00 0.00 0.00 0.15 0.00 0.34
(770) (780) (695) (655) (670) (615) (595)
(2) (0) (0) (0) (1) (0) (2)
1.17± 0.26 _+ 0.43-+ 0.61 -+ 0.45± 0.81-+ 0.84+
0.90 0.20 0.35 0.53 0.32 0.68 0.70
(9) (2) (3) (4) (3) (5) (5)
3.40 10.24 8.60 8.04 17.14
(33) (90) (54) (49) (126)
a Positive control. b SD. c N u m b e r s in parentheses are the actual numbers of colonies scored, 5 and 3 plates for each dose for nuclear and 'petite' mutations, respectively. d Locus-specific revertants.
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Discussion
The aporphine alkaloid boldine was not able to induce the SOS function as assessed by the SOS chromotest in the presence or absence of microsomal activation. In this test the rate of fl-galactosidase synthesis reflects the level of expression of SOS function involved in cell-division inhibition (suIA :: lacZ) controlled by lexA (Walker, 1985) which also controls the indirect mutagenesis (umuDC) induced by DNA-damaging agents (Bagg et al., 1981) as detected in the Ames test. Consequently, this non-induction of SOS function in the SOS chromotest implies that this alkaloid does not have genotoxic and probably mutagenic effects. These results can be correlated to the absence of mutagenic response observed with the Ames strains TA100 and TA98 which detect base-pair change and frameshift mutation, respectively. The same result was also found in the eukaryotic system S. cerevisiae (BM7126-9A strain) which can detect point and frameshift mutations. Negative results with and without microsomal activation were also obtained in strain TA102. Recently, Nozaka et al. (1990) using the Ames test showed that aporphine alkaloids are not mutagenic without $9 mix but are converted to ultimate mutagens by metabolic activation. How-
ever, when these types of alkaloids present any substituents at C9-C10 in the D ring, they were weakly mutagenic or not mutagenic. This seems to be the case for the boldine alkaloid. Boldine is, however, able to induce mitotic recombinational events in XS2316 yeast diploid cells of S. cerevisiae (Table 3). It was observed that this alkaloid induced a higher rate of gene conversion than mitotic crossing-over and this response is more pronounced in cells treated in the exponential phase of growth. It is important to notice that this growth-phase dependence is not observed for the lethal effect of boldine (data not shown). It discards, as already demonstrated for a variety of other compounds (Parry et al., 1976; Melo et al., 1986), the possible correlation between genotoxic effect and changes in the cell walls of yeast in different growth phases. However, considering that exponentially growing cells have a high level of yeast diploid cytochrome P-450 metabolic activation (Del Carretore et al., 1983; Zimmermann et al., 1984), it can be suggested that boldine does not have direct recombinogenic activity. All these observations indicate clearly that the bold±he alkaloid is not mutagenic in prokaryotic and eukaryotic organisms whereas it is recombinogenic in the S. cerevisiae cells. In addition, it is known that substances which are able to produce chro-
TABLE 3 I N D U C T I O N O F C R O S S I N G - O V E R ( + / c y h 2 ) A N D G E N E C O N V E R S I O N (leul-1/leul-2) IN D I P L O I D S T R A I N XS2316 OF Saccharomyces cerevisiae A F T E R B O L D I N E T R E A T M E N T IN T H E E X P O N E N T I A L P H A S E O F G R O W T H Agent
Dose ( k J / m 2)
8-MOP a + UVA
0 2.0
Boldine
Concentration (/~g/ml) 0 100 125 150 175 200
Survival (%)
Crossing-over (per 10 s survivors)
Gene conversion (per 10 5 survivors)
100 (932) 5 100 (934)
123.4+__15.315(115) c 399.35-20.85 (373)
14.7+ 7.02(138) 80.5+10.50 (771)
100 98 99 112 92 86
137.5+20.10 222.3±22.75 244.7±21.16 250.9+_ 38.47 215.1± 21.80 273.1+46.87
12.5+ 7.26 78.3+27.53 54.8±21.57 59.5 ± 20.81 76.6 + 27.59 45.1±13.22
(960) (940) (948) (1076) (888) (824)
(132) (209) (232) (270) (191) (225)
a Positive control. b SD. c N u m b e r s in parentheses are the actual numbers of colonies scored, 3 plates for each dose. * Genetic frequencies significant at the 1% level. ** Genetic frequencies significant at the 2% level. *** Genetic frequencies significant at the 5% level.
*** * * * **
(12) (72) (52) (64) (68) (40)
** *** ** *** **
151 m o s o m a l r e a r r a n g e m e n t s a n d exchanges with high efficiency should also b e able to alter the expression of cellular oncogenes o r activate a h i t h e r t o recessive (silent) m u t a n t allele that m a y b e involved in various stages of t u m o r d e v e l o p m e n t (Yunis, 1983). This is i m p o r t a n t m a i n l y for those early proliferative changes referred to as p r o m o tion that c a n be caused b y agents k n o w n n o t to b e m u t a g e n s in the classical sense (Yunis, 1983; L a n d et al., 1983). O n the o t h e r h a n d , in h a p l o i d yeast cells b o l d i n e was also able to i n d u c e significantly the cytop l a s m i c ' p e t i t e ' m u t a t i o n ( T a b l e 2). If, as has b e e n p r e v i o u s l y discussed (Wilkie a n d Evans, 1982; H a d l e r et al., 1983; F e r g u s o n , 1985), ' p e t i t e ' m u t a g e n e s i s correlates with carcinogenesis, then this w o u l d also suggest that the b o l d i n e a l k a l o i d m i g h t b e carcinogenic. A l t h o u g h our results m a y i n d i c a t e that this a l k a l o i d p r o b a b l y carries a l o n g - t e r m genetic risk, further s u p p o r t is necessary to decide w h e t h e r b o l d i n e a n d all the p h y t o t h e r a p e u t i c agents in which it is p r e s e n t can b e used w i t h o u t h a z a r d to h u m a n health. F u r t h e r m o r e , it seems to b e worthwhile to e x a m i n e the m u t a g e n i c a n d r e c o m b i n o g e n i c r e s p o n s e of o t h e r a l k a l o i d s that b e l o n g to the a p o r p h i n e a l k a l o i d group.
Acknowledgements W e are grateful to E. Scheidt for his assistance. W e t h a n k Dr. M. Brendel for h e l p f u l discussion. This w o r k was s u p p o r t e d b y the following Brazilian agencies: C o n s e l h h o N a c i o n a l d o Des e n v o l v i m e n t o Cientifico e T e c n o l r g i c o ( C N P q ) , P r o - R e i t o r i a de Pesquisa e P 6 s - G r a d u a ~ a o d a U F R G S , F i n a n c i a d o r a de E s t u d o s e Projetos ( F I N E P ) a n d F u n d a ~ o de A m p a r o ~t Pesquisa d o E s t a d o d o R i o G r a n d e d o Sul ( F A P E R G S ) .
References Ames, B.J., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. Bagg, A., C.J. Kenyon and G.C. Walker (1981) Inductibility of a gene product required for UV and chemical mutagenesis in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5749-5753.
Bankman, M., and M. Brendel (1989) Molecular dosimetry of 8-MOP+ UVA-induced DNA photoadducts in Saccharomyces cerevisiae: correlation of lesion number with genotoxic potential, J. Photochem. Photobiol. B Biol., 4, 57-74. Beljanski, M., and M.S. Beljanski (1982) Selective inhibition of in vitro synthesis of cancer DNA by alkaloids of the fl-carboline class, Exp. Cell. Biol., 50, 79. Culbertson, M.R., L. Charnas, M.T. Johnson and G.R. Fink (1977) Frameshift and frameshift suppressions in S. cerevisiae, Genetics, 86, 745-764. Davies, P.J., W.E. Evans and J.M. Parry (1975) Mitotic recombination induced by chemical and physical agents in yeast Saccharomyces cereoisiae, Mutation Res., 29, 301-314. Del Carretore, R., G. Bronzetti, C. Bauer, C. Corsi, R. Nieri, M. Paolini and P. Giagoni (1983) Cytochrome P-450 factors determining synthesis in strain D7 Saccharomyces cerevisiae, Mutation Res., 121, 117-123. Duke, J.A. (1985) CRC Handbook of Medicinal Herbs, CRC Press, Boca Raton, FL, pp. 358-359. Eckard, F., and R. Haynes (1977) Kinetics of mutation induction by ultraviolet light in excision-deficient yeast, Genetics, 85, 225-247. Fahrig, R. (1979) Evidence that induction and suppression of mutations and recombinations by chemical mutagens in S. cereoisiae during mitosis are jointly correlated, Mol. Gen. Genet., 168, 125-139. Ferguson, L.R. (1985) 'Petite' mutagenesis by benzidine, DAT, DAB and CDA in Saccharomyces cereoisiae, in: J.M. Parry and C.F. Arlett (Eds.), Comparative Genetic Toxicology, VCH, New York, pp. 195-203. Gaber, R.F., and M.R. Culbertson (1982) Frameshift suppression in S. cerevisiae. IV. New suppressors among spontaneous co-revertants of the group II HIS4-260 and LEU 2-3 frameshift mutations, Genetics, 101, 345-367. Guinaudeau, H., M. Leboeuf and A. Cav6 (1983) Aporphinoid alkaloids, III, J. Nat. Proc., 46, 761-835. Guinaudeu, H., M. Leboeuf and A. Cav6 (1988) Aporphinoid alkaloids, IV, J. Nat. Prod., 51,389-474. Hadler, H.I., B. Dimitrijevic and R. Mahalingam (1983) Mitochondrial DNA repair genes from normal rat liver have a common sequence, Proc. Natl. Acad. Sci. (U.S.A.), 80, 6495-6499. Henriques, J.A.P., E.J. Vicente, K.V.C.L. da Silva and A.C.G. Schenberg (1989) PSO4: a novel gene involved in errorprone repair in S. cerevisiae, Mutation Res., 218, 111-124. Jatimiliansky, J.R., and E.M. Sivori (1969) Inhibition of Scenedesmus obliquus by alkaloids, An. Soc. Cient. Argent., 187, 49-57. Kinsella, A., and N. Radman (1978) Tumor promoter induces sister chromatid exchanges: relevance to mechanisms of carcinogenesis, Proc. Natl. Acad. Sci (U.S.A.), 75, 61496153. Kunz, B., M.A. Hannan and H. Haynes (1980) Effect of tumor promoters on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae, Cancer Res., 40, 2323-2329. Land, H., L.F. Parado and R.A. Weinberg (1983) Cellular
152 oncogenes and multistep carcinogenesis, Science, 222, 771778. Lewin, D.E., M. Hollstein, M.F. Christman, E.A. Schwiers and B.N. Ames (1982) A new Salmonella tester strain (TA102) with A.T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. (U.S.A,), 79, 7445-7449. Machida, I., and S. Nakai (1980) Induction of spontaneous and UV-induced mutations during commitment to meiosis in Saccharomyces cerevisiae, Mutation Res., 73, 59-68. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mathison, L., and R. Culbertson (1985) Suppressible and non suppressible + 1 G.C base pair insertions induced by 1CR170 at the his4 locus in Saccharomyces cereoisiae, Mol. Cell. Biol., 5, 2247-2256. McCarm, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Melo, A.A., C.B. Querol, A.T. Henriques and J.A.P. Henriques (1986) Cytostatic, cytotoxic and mutagenic effects of voacristine, an indole alkaloid, in wild-type and repair deficient yeasts, Mutation Res., 171, 17-24. Miller, E.C., and J.A. Miller (1981) Mechanisms of chemical carcinogenesis, Cancer, 47, 1055-1064. Moustacchi, E., V. Favaudon and E. Bisagni (1983) Likelihood of the new antitumoral drug 10(a-dimethylaminopropylamino)-6-methyl-5H-pyrido[3',4' : 4,5]pyrrolo[2,3-g]isoquinoline (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand breaks in vivo and its non mutagenicity in yeast, Cancer Res., 43, 3700-3706. Neumeyer, J.L. (1985) Synthesis and structure-activity relationship of aporphines as dopamine receptor agonist and antagonist, in: S.D. Phillipson, M.F. Roberts and M.H. Zenk (Eds.), Chemistry and Biology of Isoquinoline Alkaloids, Springer, Berlin, pp. 146-170.
Nozaka, T., F. Watanabe, S. Tadaki, M. Ishino, I. Morimoto, J. Kunitomo, H. Ishii and S. Natori (1990) Mutagenicity of isoquinoline alkaloids, specially of the aporphine type, Mutation Res., 240, 267-279. Ogur, R., R. St. John and S. Nagai (1957) Tetrazolium overlay technique for population studies of respiration deficiency of yeast, Science, 125, 928-929. Parry, I.M., P.J. Davies and W.E. Evans (1976) The effects of 'cell age' upon the lethal effects of physical and chemical mutagens in the yeast Saccharomyces cerevisiae, Mol. Gen. Genet., 146, 27-35. Quillardet, P., and M. Hofnung (1985) The SOS Chromotest, colorimetric bacterial assay for genotoxins: Procedures, Mutation Res., 147, 65-78. Quillardet, P., C. Bellecombe and M. Hofnung (1985) The SOS Chromotest, colorimetric bacterial assay for genotoxins: validation study with 83 compounds, Mutation Res., 147, 79-85. Von Poser, G.L., H.H.R. Andrade, K.V.C.L. Da Silva, A.T. Henriques and J.A.P. Henriques (1990) Genotoxic, mutagenic and recombinogenic effects of Rauwolfia alkaloids, Mutation Res., 232, 37-43. Walker, G.C. (1985) Inducible DNA repair systems, Annu. Rev. Biochem., 54, 425-457. Wilkie, D., and I.H. Evans (1982) Mitochondria and yeast cell surface: implications for carcinogenesis, Trends Biochem. Sci., 7, 147-151. Yunis, J.J. (1983) The chromosomal basis of human neoplasia, Science, 221,227-236. Zimmermann, F.K., R.C. yon Borstel, E.S. yon Halle, J.M. Parry, D. Siebert, G. Zeiterberg, R. Barle and H. Loprieno (1984) Testing of chemicals for genetic activity with Saccharornyces cerevisiae: a report of the US Environmental Protection Agency Gene-Tox Program, Mutation Res., 133, 199-244.
145
MUTGEN 01652
Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms Paulo Roberto H. Moreno 1, Vera Maria F. Vargas 2, Heloisa Helena R. Andrade 3, Amelia T. Henriques 1 and Jogo Antonio P. Henriques 4 I Curso de Pds-Gradua~o em Farmdcia-UFRGS, 2 Departamento do Meio Ambiente, Secretaria da Saftde e do Meio Ambiente, Departamento de Fisiologia, Farmacologia e Biofisica and 4 Departamento de Gendtica, Instituto de Bioci~ncias-UFRGS, Porto Alegre, R S (Brazil) (Received 17 July 1990) (Revision received 2 November 1990) (Accepted 7 November 1990)
Keywords: Boldine; Ames test; SOS chromotest; Yeast
Summary The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.
Alkaloids are a large group of natural products recognized for their remarkable pharmacological activities. Among the different classes of alkaloids the aporphines have been pharmacologically studied with regard to their properties in the central nervous system (CNS) as dopamine agonists (Neumeyer, 1985). Aporphines have been used as 'rigid' analogues of dopamine in the determination of the interaction with the dopaminergic receptors (Neumeyer, 1985).
Correspondence: Dr. J.A. P~gas Henriques, Instituto de Bioci8ncias-UFRGS, Setor de Biofisica - BIO-3, Rua Sarmento Leite 500, 90049 Porto Alegre, RS (Brazil).
Some aporphine alkaloids could inhibit the growth of the microorganisms Scenedesmus obliquus (Jatimiliansky and Sivori, 1969). Boldine is an aporphine alkaloid usually found in the plant families Magnoliaceae, Annonaceae, Rhamnaceae and Monimiaceae (Guinaudeau et al., 1983, 1988). In the last one it has the species Peumus boldus (boldo-do-Chile), widely used in folk medicine all over the world (Duke, 1985). In human therapy preparations of medicinal plants are widely used although little information on their potential risk to health is available. It has been suggested that alkaloids can be considered potentially mutagenic, since the therapeutic action of certain alkaloids is related to its interaction with DNA (Beljansky and Beljansky, 1982;
146
Moustacchi et al., 1983; Melo et al., 1986; Von Poser et al., 1990) and the mutagenic and carcinogenic effects are due to this interaction (Miller and Miller, 1981). Therefore, it is yery important to ascertain if boldine, as a model of aporphine alkaloids, exhibits genotoxic and mutagenic activities in prokaryotic and eukaryotic cells. This is important for long-term risk estimates since 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 and mutagenic effects of boldine by means of the SOS chromotest (Quillardet and Hofnung, 1985) and the Ames test (Ames et al., 1975). Several studies have indicated that most chemical mutagens are potential inducers of mitotic recombination (Davies et al., 1975; Fahrig, 1979; Kunz et al., 1980; Von Poser et al., 1990). 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 Radmann, 1978) or in a cocarcinogenic process (Kunz et al., 1980). Thus, the diploid strain of Saccharomyces cereoisiae XS2316 (Machida and Nakai, 1980), which allows the detection of the 2 forms of mitotic recombination (crossing-over and gene conversion), was employed to determine the possible recombinogenic activities of this alkaloid in a eukaryotic system. The boldine alkaloid was also tested for induction of 2 types of locus-specific mutation in haploid yeast cells, reversion of the lysl-1 ochre and his4-38 frameshift alleles. Moreover, boldine was tested for its ability to induce mitochondrial 'petite' mutations. Materials and methods
Strains Escherichia coli SOS-chromotest tester strain PQ37 (as described by QuiUardet and Hofnung, 1985) was obtained from Dr. P. Qnillardet. The Salmonella typhimurium strains TA98, TA100 and TA102, as described by Maron and Ames (1983), were kindly provided by Dr. B.N. Ames. The haploid strain of Saccharomyces cereoisiae BM7126-9A (a leul-I ade2-1 his4-38 leu2-3 ura rho °) was constructed by Bankmann and Brendel
4
CH30
~
NCH 3 "H
(
c%o,
OH
BOLDINE
Fig. 1. Chemical structure of the aporphine alkaloid boldine.
(1989). The N123 (a his1 ) haploid yeast strain was kindly provided by Dr. E. Moustacchi. The diploid S. cereuisiae strain XS2316 has the following genotype (Machida and Nakai, 1980): + leul-1 trp5-48 + + + a his1-1 ade6 ° leul-12 + cyh2 m e t l 3 lys5-1 a his1-1
Chemical products The aporphine alkaloid boldine (Fig. 1) was obtained from Sigma Co. (St. Louis, MO, U.S.A.). Its purity level was confirmed by 2 different chromatographic systems. To carry out the SOS chromotest and the Ames test the alkaloid was diluted in spectrophotometric-grade dimethyl sulfoxide (DMSO). For treatment of yeast cells a stock solution of 1 mg/mi boldine was prepared using the mixture saline solution (0.8 ml) and absolute ethanol (0.2 ml) immediately prior to use. The appropriate solvent controls included in the genetic tests were found to be negative. 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 methionine and SC - adenine) were a series of SC media in which one of the amino acids or bases had been omitted. The cycloheximide medium (SC + cyh) was SC supplemented with 200/xg cycloheximide (Calbiochem, U.S.A.) per 100 ml SC.
147
For mutagenesis SC medium was MM supplemented with 3 mg each of lysine and leucine, 2 mg each of arginine, uracil, methionine, histidine, and tryptophan and 0.5 mg of adenine, per 100 ml MM. Omission media lacking lysine, histidine were sub-supplemented with 0.1 mg of lysine or histidine, respectively, per 100 ml omission medium. LB medium contained 1% bactopeptone (Difco), 0.5% yeast extract (Difco) and 1% NaC1. S O S chromotest The SOS chromotest was performed according to Quillardet et al. (1985). An exponential-phase culture of PQ37, grown in LB medium plus ampicillin (20 #g/ml) at 37°C, was diluted 1:10 into fresh medium or in mixture of $9 mix for metaboric activation. Fractions (0.6 ml) were distributed into glass test tubes containing 20 #1 of boldine. After 2-h incubation at 37 °C with shaking, 0.3-ml samples were used to assay /3-galactosidase and alkaline-phosphatase activities. SOS induction factor in treated cells was obtained from the ratio of /3-galactosidase and alkaline phosphatase activities, compared to untreated cells. As positive controls, 4-nitroquinoline 1-oxide (4NQO) and aflatoxin B1 (AFB1) (both from Sigma) were used for the tests without and with metabolic activation, respectively. Ames test Mutagenicity was assayed by the spot test procedure (Ames et al., 1975). Samples of boldine (0.5, 5, 50, 100 and 200/~g/plate) were spotted on a plate with 0.1 ml of tester bacterial cultures in the presence or absence of $9 mix (20 #1 of $9 fraction for 500/~1 of $9 mix) and incubated in the dark at 37°C for 48 li. For strain TA102 the mutagenicity was assayed by the preincubation procedure (Maron and Ames, 1983). Negative (DMSO) and positive (5 #g sodium azide per plate for TA100, 0.5 fig 4NQO per plate for TA98 and 100 /~g hydrogen peroxide for TA102) controls were included in each assay. 2-Aminoanthracene (2-AA, 0.1 txg per plate) for TA102 and AFB 1 (0.5 /~g per plate) for TA100 and TA98 were used as positive controls for the metabolic assays. Microsomal fraction The microsomal fraction $9 was prepared from livers of Sprague-Dawley rats pretreated with
polychlorinated biphenyl mixture (Aroclor 1254) as described by Maron and Ames (1983). The $9 mix metabolic activation mixture was prepared according to Quillardet and Hofnung (1985) for the SOS chromotest and according to Maron and Ames (1983) for the Ames test. Yeast growth conditions Cells were routinely cultured in YEPD from single-colony isolates. In each independent experiment, 2 X 106 cells/ml were seeded and shaken vigorously at 30 °C for 3 days to obtain cultures in the stationary phase of growth. Exponentialphase cultures were obtained by inoculation of 5 x 105 cells of the same stationary YEPD culture in 5 ml of YEPD medium. After 12 h of incubation in the same conditions, the cultures contained 2 x 107 cells/ml. Cells were harvested and washed twice with saline (0.9% NaC1). Clumps were dissociated by sonication of the cell suspension for 15 s in a Thornton ultrasonic disintegrator. The cell number and the percentage of budding cells were determined with a counting chamber. Detection of boldine-induced reverse mutation A suspension of cells (2 X 108 cells/ml) in exponential growth phase were incubated for 20 h at 30 ° C with various concentrations of boldine. After treatment, survival (on SC, 5-7 days, 30 ° C) and LYS or HIS prototrophic colonies (on appropriate omission media, 7-10 days, 30°C) were scored. HIS prototrophic revertants were picked onto SC - his medium and after 2 days at 30 ° C replicated onto S C - l e u medium. After 3 days at 30°C co-reversion to LEU prototrophy was scored. Both lysl-1 and ade2-1 are suppressible UAA (ochre) nonsense alleles (Eckardt and Haynes, 1977). Therefore, white-colored LYS-prototrophic mutants are, with high probability, locus-non-specific intergenic revertants (suppressors) whereas redcolored LYS-prototrophic colonies demonstrate locus-specific mutational events. Both his4-38 and leu2-3 alleles represent frameshift mutations (his438: + G C insertion) (Mathison and Culbertson, 1985) induced after treatment with ICR-170 (Culbertson et al., 1977) and are suppressible by group-II frameshift suppressors (Gaber and Culbertson, 1982). Thus, double prototrophic mutants induced in his4-38 leu2-3 strains can be referred
148 a n d i n c u b a t e d for 7 - 8 d a y s at 3 0 ° C . Colonies g r o w n o n S C m e d i u m y i e l d e d d a t a of cell survival a n d colonies g r o w n o n S C - leu a n d SC + cyh were scored for i n t r a g e n i c r e c o m b i n a t i o n (gene conversion) a n d i n t e r g e n i c r e c o m b i n a t i o n (crossing-over), respectively. I n o r d e r to m e a s u r e the exact f r e q u e n c y of r e c i p r o c a l crossing-over, it is necessary to e l i m i n a t e the p o s s i b i l i t y t h a t some c y c l o h e x i m i d e - r e s i s t a n t colonies h a d resulted f r o m reversion at the C Y H 2 locus, as well as m o n o s o m y on c h r o m o s o m e VII. F o r this p u r p o s e , the cycloh e x i m i d e - r e s i s t a n t colonies were r e p l i c a - p l a t e d on a series of m e d i a , S C - lys, S C - m e t a n d S C ade, for screening these l i n k e d m a r k e r s of cyh2. T h e e x p e r i m e n t was r e p e a t e d at least 3 times. Platings were d o n e in t r i p l i c a t e for each dose so t h a t a m i n i m u m o f 200 survivors a n d of 7 0 - 9 0 r e c o m b i n a n t s p e r p o i n t were scored.
to as suppressors whereas leu-auxotrophic H I S revertants ( a n d vice versa) r e p r e s e n t locus-specific revertants. T h e e x p e r i m e n t was r e p e a t e d at least 3 times.
Detection of cytoplasmic "petite' mutation T h e frequency of m i t o c h o n d r i a l r e s p i r a t o r y - d e ficient m u t a n t s or ' p e t i t e s ' was d e t e r m i n e d b y the t r i p h e n y l t e t r a z o l i u m overlay t e c h n i q u e ( O g u r et al., 1957).
Detection of induced mitotic recombination Suspensions of ceils in s t a t i o n a r y (2 × 10 8 c e l l s / m l ) a n d e x p o n e n t i a l p h a s e s (2 x 10 7 c e l l s / m l ) of g r o w t h were i n c u b a t e d for 20 h at 30 o C with various c o n c e n t r a t i o n s of b o l d i n e . A f t e r treatment, the cells were d i l u t e d in saline, p l a t e d o n 3 kinds of m e d i u m (SC, SC - leu a n d SC + cyh)
TABLE 1 SOS FUNCTION-INDUCING ACTIVITY OF BOLDINE IN Escherichia coli STRAIN PQ37 IN THE SOS CHROMOTEST Substance
Concentration (ng/test)
fl-Galactosidase(B) (units)
Alkaline phosphatase (P) (units)
B
Induction factor
Without metabolicactivation 4-NQO a
0 10 100
Boldine
0 5 1 5 1 5 1
×10 ~ x 102 x 102 x 103 x 103 x 104
109 347 1747
509 509 438
0.214 0.681 4.063
1 3.18 18.98
109 133 145 116 116 118 109
509 500 500 525 519 531 540
0.214 0.266 0.290 0.221 0.224 0.222 0.202
1 1.24 1.36 1.03 1.05 1.04 0.94
167 443 1018
1578 1629 1617
0.106 0.272 0.629
1 2.57 5.93
230 200 169 193 174 191 165
952 937 950 952 1020 990 984
0.230 0.213 0.178 0.203 0.171 0.193 0.168
1 0.93 0.77 0.88 0.74 0.84 0.73
With metabolicactivation AFB1 a
0 5 20
Boldine
a Positive controls.
0 5 x 102 1 x 103 2.5 x 10 3 5 × 103 7.5 x 103 1 × 104
149
8-Methoxypsoralen + UVA treatment 8-Methoxypsoralen (8-MOP) (Sigma) was used as positive control in the determination of mutational and recombinational events in yeast. The conditions were as previously described by Henriques et al. (1989). Results
Table 1 shows that boldine, in contrast to data obtained for positive controls, clearly did not induce SOS function (fl-galactosidase synthesis), when tested with the SOS chromotest with and without metabolic activation. These results suggest that this alkaloid does not have genotoxic activity. To search for mutagenicity of boldine we used the haploid yeast strain BM7126-9A which allows the simultaneous scoring of locus-specific mutation in 2 genes: point mutation in the ochre allele lysl-1 and frameshift mutation in the his4-38 allele; locus specificity of reversion is discriminated for by detection of intergenic suppression (co-reversion of ade2-1 for ochre suppression and of leu2-3 for group-II frameshift suppression). Table 2 shows that at the doses applied boldine was non-toxic and was not able to change significantly the frequency of reversion of both lysl-1 and
his4-38 alleles in comparison with the non-treated control. The same lack of mutagenic activity has also been found in the Ames test using strains TA100 (base-pair substitution), TA98 (frameshift mutation) and TA102 (detects oxidative and alkylating mutagens and active forms of oxygen; Lewin et al., 1982) with and without microsomal activation (data not shown). It can be also observed in Table 2 that the mitochondrial 'petite' mutation is significantly induced by the aporphine alkaloid boldine. The search for a recombinogenic effect of this alkaloid was carried out in stationary and exponential cultures of yeast diploid cells. Table 3 shows that at the doses applied the frequencies of convertants (LEU +) and recombinants (CYH +) induced by boldine were significantly increased (Student's t-test). The induction of gene conversion in the exponentially growing diploid yeast cells was higher than crossing-over. In fact it corresponds to a 5-6-fold increase of convertogenic events over the spontaneous background. Although the frequency of both convertants and recombinants in stationary diploid cells was less pronounced to that observed in exponential-phase cells, both induction kinetics were practically the same (data not shown).
TABLE 2 I N D U C T I O N OF F R A M E S H I F T (his4-38), P O I N T M U T A T I O N (lysl-1, O C H R E A L L E L E ) A N D C Y T O P L A S M I C 'PETITE' M U T A T I O N S IN H A P L O I D YEAST STRAINS, A F T E R B O L D I N E T R E A T M E N T Agent
8-MOP a + UVA Boldine
Dose
Concentration
Survival (Z)
(kJ//m2)
(ttg/ml)
N123
0 2.0
-
-
0 25 50 100 125 150 200
100 94 75 74 77
(933) (879) (696) (693) (735)
his4/lO 7 survivors d
l y s l / l O 7 survivors d
'Petite'
BM7126-9A
BM7126-9A
BM7126-9A
m u t a n t s (%) N123
100(672) 18 (273)
0.29+0.22 b (2) c 1.0 +0.37 (27)
1.80_+ 0.72 (16) 115.13_+10.61 (188)
100 101 90 85 87 80 77
0.26 0.00 0.00 0.00 0.15 0.00 0.34
(770) (780) (695) (655) (670) (615) (595)
(2) (0) (0) (0) (1) (0) (2)
1.17± 0.26 _+ 0.43-+ 0.61 -+ 0.45± 0.81-+ 0.84+
0.90 0.20 0.35 0.53 0.32 0.68 0.70
(9) (2) (3) (4) (3) (5) (5)
3.40 10.24 8.60 8.04 17.14
(33) (90) (54) (49) (126)
a Positive control. b SD. c N u m b e r s in parentheses are the actual numbers of colonies scored, 5 and 3 plates for each dose for nuclear and 'petite' mutations, respectively. d Locus-specific revertants.
150
Discussion
The aporphine alkaloid boldine was not able to induce the SOS function as assessed by the SOS chromotest in the presence or absence of microsomal activation. In this test the rate of fl-galactosidase synthesis reflects the level of expression of SOS function involved in cell-division inhibition (suIA :: lacZ) controlled by lexA (Walker, 1985) which also controls the indirect mutagenesis (umuDC) induced by DNA-damaging agents (Bagg et al., 1981) as detected in the Ames test. Consequently, this non-induction of SOS function in the SOS chromotest implies that this alkaloid does not have genotoxic and probably mutagenic effects. These results can be correlated to the absence of mutagenic response observed with the Ames strains TA100 and TA98 which detect base-pair change and frameshift mutation, respectively. The same result was also found in the eukaryotic system S. cerevisiae (BM7126-9A strain) which can detect point and frameshift mutations. Negative results with and without microsomal activation were also obtained in strain TA102. Recently, Nozaka et al. (1990) using the Ames test showed that aporphine alkaloids are not mutagenic without $9 mix but are converted to ultimate mutagens by metabolic activation. How-
ever, when these types of alkaloids present any substituents at C9-C10 in the D ring, they were weakly mutagenic or not mutagenic. This seems to be the case for the boldine alkaloid. Boldine is, however, able to induce mitotic recombinational events in XS2316 yeast diploid cells of S. cerevisiae (Table 3). It was observed that this alkaloid induced a higher rate of gene conversion than mitotic crossing-over and this response is more pronounced in cells treated in the exponential phase of growth. It is important to notice that this growth-phase dependence is not observed for the lethal effect of boldine (data not shown). It discards, as already demonstrated for a variety of other compounds (Parry et al., 1976; Melo et al., 1986), the possible correlation between genotoxic effect and changes in the cell walls of yeast in different growth phases. However, considering that exponentially growing cells have a high level of yeast diploid cytochrome P-450 metabolic activation (Del Carretore et al., 1983; Zimmermann et al., 1984), it can be suggested that boldine does not have direct recombinogenic activity. All these observations indicate clearly that the bold±he alkaloid is not mutagenic in prokaryotic and eukaryotic organisms whereas it is recombinogenic in the S. cerevisiae cells. In addition, it is known that substances which are able to produce chro-
TABLE 3 I N D U C T I O N O F C R O S S I N G - O V E R ( + / c y h 2 ) A N D G E N E C O N V E R S I O N (leul-1/leul-2) IN D I P L O I D S T R A I N XS2316 OF Saccharomyces cerevisiae A F T E R B O L D I N E T R E A T M E N T IN T H E E X P O N E N T I A L P H A S E O F G R O W T H Agent
Dose ( k J / m 2)
8-MOP a + UVA
0 2.0
Boldine
Concentration (/~g/ml) 0 100 125 150 175 200
Survival (%)
Crossing-over (per 10 s survivors)
Gene conversion (per 10 5 survivors)
100 (932) 5 100 (934)
123.4+__15.315(115) c 399.35-20.85 (373)
14.7+ 7.02(138) 80.5+10.50 (771)
100 98 99 112 92 86
137.5+20.10 222.3±22.75 244.7±21.16 250.9+_ 38.47 215.1± 21.80 273.1+46.87
12.5+ 7.26 78.3+27.53 54.8±21.57 59.5 ± 20.81 76.6 + 27.59 45.1±13.22
(960) (940) (948) (1076) (888) (824)
(132) (209) (232) (270) (191) (225)
a Positive control. b SD. c N u m b e r s in parentheses are the actual numbers of colonies scored, 3 plates for each dose. * Genetic frequencies significant at the 1% level. ** Genetic frequencies significant at the 2% level. *** Genetic frequencies significant at the 5% level.
*** * * * **
(12) (72) (52) (64) (68) (40)
** *** ** *** **
151 m o s o m a l r e a r r a n g e m e n t s a n d exchanges with high efficiency should also b e able to alter the expression of cellular oncogenes o r activate a h i t h e r t o recessive (silent) m u t a n t allele that m a y b e involved in various stages of t u m o r d e v e l o p m e n t (Yunis, 1983). This is i m p o r t a n t m a i n l y for those early proliferative changes referred to as p r o m o tion that c a n be caused b y agents k n o w n n o t to b e m u t a g e n s in the classical sense (Yunis, 1983; L a n d et al., 1983). O n the o t h e r h a n d , in h a p l o i d yeast cells b o l d i n e was also able to i n d u c e significantly the cytop l a s m i c ' p e t i t e ' m u t a t i o n ( T a b l e 2). If, as has b e e n p r e v i o u s l y discussed (Wilkie a n d Evans, 1982; H a d l e r et al., 1983; F e r g u s o n , 1985), ' p e t i t e ' m u t a g e n e s i s correlates with carcinogenesis, then this w o u l d also suggest that the b o l d i n e a l k a l o i d m i g h t b e carcinogenic. A l t h o u g h our results m a y i n d i c a t e that this a l k a l o i d p r o b a b l y carries a l o n g - t e r m genetic risk, further s u p p o r t is necessary to decide w h e t h e r b o l d i n e a n d all the p h y t o t h e r a p e u t i c agents in which it is p r e s e n t can b e used w i t h o u t h a z a r d to h u m a n health. F u r t h e r m o r e , it seems to b e worthwhile to e x a m i n e the m u t a g e n i c a n d r e c o m b i n o g e n i c r e s p o n s e of o t h e r a l k a l o i d s that b e l o n g to the a p o r p h i n e a l k a l o i d group.
Acknowledgements W e are grateful to E. Scheidt for his assistance. W e t h a n k Dr. M. Brendel for h e l p f u l discussion. This w o r k was s u p p o r t e d b y the following Brazilian agencies: C o n s e l h h o N a c i o n a l d o Des e n v o l v i m e n t o Cientifico e T e c n o l r g i c o ( C N P q ) , P r o - R e i t o r i a de Pesquisa e P 6 s - G r a d u a ~ a o d a U F R G S , F i n a n c i a d o r a de E s t u d o s e Projetos ( F I N E P ) a n d F u n d a ~ o de A m p a r o ~t Pesquisa d o E s t a d o d o R i o G r a n d e d o Sul ( F A P E R G S ) .
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