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Genotoxic effects of structurally related β-carboline alkaloids
Mutation Research 379 Ž1997. 135–149
Genotoxic effects of structurally related b-carboline alkaloids Jaqueline N. Picada a,b, Katia V.C.L. da Silva b, Bernardo Erdtmann c , Amelia ´ T. Henriques a, Joao ˜ A.P. Henriques b,) a
b
Curso de Pos-Graduac ¸ao Farmaceuticas e Departamento de Produc¸ao ´ ˜ em Ciencias ˆ ˆ ˜ de Materia ´ Prima da Faculdade de Farmacia, ´ UFRGS, AÕ. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil Departamento de Biofısica e Centro de Biotecnologia, UFRGS, AÕ. Bento Gonc¸alÕes 9500, predio ´ ´ 43421, Campus do Vale, Caixa Postal 15005, CEP 91501-970, Porto Alegre, RS, Brazil c Departamento de Genetica, UFRGS, AÕ. Bento Gonc¸alÕes, 9500, predio ´ ´ 43323, Campus do Vale, CEP 91501-970, Porto Alegre, RS, Brazil Received 17 January 1997; revised 29 April 1997; accepted 2 May 1997
Abstract b-Carboline alkaloids, found in medicinal plants, tobacco smoke and well-cooked foods, have shown a variety of actions in biological systems related to their interaction with DNA. Therefore, these alkaloids can be considered potentially mutagenic. In this work, the genotoxic, mutagenic, and cytotoxic activities of three aromatic b-carboline alkaloids Žharman, harmine, and harmol. and two dihydro-b-carboline alkaloids Žharmaline and harmalol. were evaluated by means of the Salmonellarmicrosome assay Ž Salmonella typhimurium TA98, TA97, TA100, and TA102. and SOS chromotest Ž Escherichia coli PQ37. with and without metabolic activation. Moreover, harman and harmine were analyzed by the micronucleus assay in vivo. It was shown that genotoxicity was inhibited by the addition of S9 mix for aromatic b-carbolines harman and harmol in TA97. However, harmine showed signs of mutagenicity only in the presence of S9 mix in TA98 and TA97 frameshift strains. In the SOS chromotest, only harman induced SOS functions in the absence of S9 mix. Dihydro-b-carbolines were not genotoxic in any of the microorganisms used. The negative responses obtained in the micronucleus assay indicated that harman and harmine were not able to induce chromosomal mutations. q 1997 Elsevier Science B.V. Keywords: b-Carboline; Genotoxicity; Micronucleus assay; Mutagenicity; Salmonellarmicrosome assay; SOS chromotest
1. Introduction b-Carboline alkaloids, also known as harmala’s alkaloids because they were first isolated from Peganum harmala ŽZygophillaceae., are natural products that have been consumed as hallucinogenic drinks, snuffs and other forms in the Amazon basin, ) Corresponding author. Fax: q55 Ž51. 336-2779; e-mail: [email protected]
where they are found in Banisteriopsis spp. and Tetrapteris spp. ŽMalpighiaceae., Virola spp. ŽMyristicaceae. and Anadenanthera spp. ŽLeguminosae. w1,2x. Passiflora spp. ŽPassifloraceae. Žpopularly know as Maracujas ´ ., which contains b-carboline alkaloids, such as harman, is one of the plants widely used in medical practice in Brazil, mainly for its anti-spasmodic and sedative properties w1x. In addition to their natural occurrence in plants, bcarbolines have been found in a number of other
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 1 6 - 4
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J.N. Picada et al.r Mutation Research 379 (1997) 135–149
sources. Harman and norharman have also been detected in tobacco smoke and well-cooked foods, reflecting their formation from tryptophan by pyrolysis w3–5x. These compounds have been of interest for some time because of the variety of actions which they evoke in biological systems w1,6,7x. Harman and norharman induce a co-mutagenic effect with a number of compounds in Salmonella typhimurium TA98 and TA100 w8–10x and in mammalian cell culture w11,12x. Both are cytotoxic and mutagenic to Chinese hamster lung cells ŽCHL cells. w13x. They are also reported to induce sister chromatid exchange ŽSCE. in human peripheral lymphocytes w14x. Treatment of mice with 0.1% harman in their diet for 4 weeks resulted in DNA adducts in the liver and kidney w15x. Their activities in prokaryotic and eukaryotic cells have been attributed in part to the ability of these chemicals to interact with DNA. Hayashi et al. w16x found that harman and norharman react with DNA by intercalation: DNA induced a quenching of the fluorescence of norharman, and a marked red shift and hypochromism of the absorption spectra of norharman and harman. Circular supercoiled DNA is unwound by norharman. Hayashi et al. w16x reported that harman intercalates more easily into DNA than norharman. Duportail and Lami w17x showed that the hydrogenation of a double bond of the pyridine ring converting harmine, an aromatic b-carboline, to harmaline, a dihydro-b-carboline, greatly alters the interaction of the molecule with DNA. Measurements of the viscosity indicated intercalation of harmine only. It is very well known that the mutagenic and carcinogenic effects of the various compounds are related to their intercalation with DNA w18,19x. So, it is very important to find out if b-carboline alkaloids show genotoxic and mutagenic activities in prokaryotic cells. Since these compounds are present in human food and induce mutagenesis in mammalian cells in culture, it is also necessary to study their mutagenicity in vivo. Thus, the genotoxic activity of three aromatic b-carboline alkaloids, harman, harmine, and harmol, and two dihydro-b-carboline alkaloids, harmaline and harmalol was evaluated by means of the Salmonellarmicrosome assay w20,21x and the SOS chromotest w22,23x. Moreover, harman and harmine were tested for their ability to induce micronuclei in mice bone marrow polychromatic erythrocytes w24,25x.
2. Materials and methods 2.1. Chemical products The structural formula and the chemical name of each b-carboline alkaloid tested for genotoxicity is shown in Fig. 1. The aromatic b-carbolines, harman ŽCAS 2165584-5; not specified purity., harmine ŽCAS 343-27-1; approx. 98% purity., and harmol ŽCAS 149022-16-2; not specified purity. hydrochlorides, and the dihydro-b-carbolines, harmaline ŽCAS 6027-98-1; approx. 95% purity. and harmalol ŽCAS 6028-07-5; approx. 90% purity. hydrochloride, were obtained from Sigma Co ŽSt. Louis, MO, USA.. To carry out the SOS chromotest, Salmonellarmicrosome assay, and micronucleus assay, harmine was dissolved in sterile distilled water while the other four alkaloids tested were dissolved in spectrophotometric-grade dimethylsulfoxide ŽDMSO. and sterile distilled water Ž1:1.. 2.2. Strains Escherichia coli SOS-chromotest tester strain PQ37, which has the following genotype: Fy, thr leu his-4 pyrD thi galE galk (galT) lac169 srl300
Fig. 1. Chemical structure of the b-carboline alkaloids. A: aromatic b-carbolines. B: dihydro-b-carbolines.
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
tut Pasteur, Paris ŽFrance.. The Salmonella typhimurium strains TA98, TA97, TA100, and TA102, as described by Maron and Ames w20x, were kindly provided by Dr. B.N. Ames, University of California, Berkeley, CA ŽUSA..
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ŽMoltox e .. The S9 metabolic activation mixture was prepared according to Quillardet and Hofnung w22x for the SOS chromotest and according to Maron and Ames w20x for the Salmonellarmicrosome assay. 2.4. SOS chromotest
2.3. Microsomal fraction The postmicrosomal fraction S9 was prepared from livers of Sprague–Dawley rats pretreated with polychlorinated biphenyl mixture ŽAroclor 1254., purchased from Molecular Toxicology Inc.
The SOS chromotest was performed according to Quillardet and Hofnung w22x. An exponential-phase culture of Escherichia coli PQ37, grown in LB medium Ž1% bacto tryptone ŽDifco., 0.5% bacto yeast extract ŽDifco. and 1% NaCl. plus ampicillin
Table 1 SOS function inducing activity of b-carboline alkaloids in E. coli PQ37 in the SOS chromotest without metabolic activation Substance 4-NQO
a
Harman
Harmine
Harmol
Harmaline
Harmalol
a
Dose Žmgrtest.
b-Galactosidase ŽB. Žunits.
Alkaline phosphatase ŽP. Žunits.
BrP
Induction factor
0 0.01 0.05 0 0.1 1 5 10 50 0 0.1 1 10 100 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 500 0 0.01 0.1 1 10
410 1225 1605 46 83 80 84 95 191 46 69 69 153 46 521 577 569 569 648 267 410 561 474 421 456 596 900 521 584 548 783 1170
1380 1670 1410 582 571 571 607 500 430 582 554 582 357 143 1530 1530 1400 1330 1130 450 1380 1740 1550 1690 1460 921 320 1530 1530 1500 1450 1600
0.297 0.733 1.138 0.079 0.145 0.140 0.138 0.190 0.444 0.079 0.125 0.119 0.429 0.322 0.341 0.377 0.406 0.428 0.573 0.593 0.297 0.322 0.306 0.249 0.312 0.647 2.813 0.341 0.382 0.365 0.540 0.731
1.00 2.47 3.83 1.00 1.84 1.77 1.75 2.41 5.62 1.00 1.58 1.51 5.43 4.08 1.00 1.11 1.19 1.26 1.68 1.74 1.00 1.08 1.03 0.84 1.05 2.18 9.47 1.00 1.12 1.07 1.58 2.14
b
Positive control. The induction factor at concentration C is defined as Ic s R crR 0 , where R c and R 0 are the ratios of b-galactosidase and alkaline phosphatase activities with and without added DNA-damaging agent. b
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
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Table 2 SOS function inducing activity of b-carboline alkaloids in E. coli PQ37 in the SOS chromotest with metabolic activation Substance AFB1
a
Harman
Harmine
Harmol
Harmaline
Harmalol
Dose Žmgrtest.
b-Galactosidase ŽB. Žunits.
Alkaline phosphatase ŽP. Žunits.
BrP
Induction factor
0 0.005 0.02 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 250 0 0.1 1 10
410 1230 1650 439 490 488 488 455 439 488 520 520 390 411 457 457 411 381 439 455 423 553 683 878 411 488 457 854
1380 1270 827 976 1105 1140 1100 793 976 1100 1070 1590 1100 1360 1300 1220 1330 1430 976 945 1070 915 854 793 1360 1250 1270 1300
0.297 0.969 1.995 0.450 0.443 0.428 0.444 0.574 0.450 0.444 0.486 0.327 0.355 0.302 0.352 0.375 0.309 0.266 0.450 0.481 0.395 0.604 0.800 1.107 0.302 0.390 0.360 0.657
1.00 3.25 6.69 1.00 0.99 0.95 0.99 1.28 1.00 0.99 1.08 0.73 0.79 1.00 1.17 1.24 1.02 0.88 1.00 1.07 0.88 1.34 1.78 2.46 1.00 1.29 1.19 2.18
b
a
Positive control. The induction factor at concentration C is defined as Ic s R crR 0 , where R c and R 0 are the ratios of b-galactosidase and alkaline phosphatase activities with and without added DNA-damaging agent. b
Ž20 mgrml. at 378C, was diluted 1:10 into fresh medium or in S9 mix for metabolic activation. Aliquots of 600 ml were distributed into glass test tubes containing 20 ml of the alkaloids to be tested. As positive controls, aflatoxin B1 ŽAFB1. and 4nitroquinoline 1-oxide Ž4-NQO. Žboth from Sigma Chemical, St. Louis, MO, USA. were used with and without metabolization. After 2 h of incubation at
378C with shaking, 300 ml samples were taken to assay for b-galactosidase and alkaline-phosphatase activities. In this assay, the sfiA gene linked bgalactosidase activity is determined as a measure of the induction of the SOS repair system. The activity of the constitutive enzyme alkaline phosphatase was used as a measure of protein synthesis and toxicity. Enzyme activities were assayed in Miller units w26x.
Notes to Table 3: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values of at least two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 194 " 25; ŽqS9. aflatoxin B1 Ž1 mgrplate., 476 " 41. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2..
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
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Table 3 Induction of Hisq revertants in S. typhimurium TA98 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance NC d Harman
Response NC Harmine
Dose
TA98 yS9
Žmgrplate.
MI
10 20 30 40 50 100 150 200 250
1.1 1.0 1.0 0.9 0.9
e
10 20 30 40 50 100 150 200 250
1.1 1.1 1.1 1.0 0.9
20 40 60 80 100
1.0 1.1 1.0 1.1 0.9
50 100 150 200 250 300 400 500
0.9 1.0 1.0 1.0 1.0
Response NC Harmalol
Response
18 " 3 20 " 4 18 " 2 17 " 1 16 " 2 16 " 1
b
%S 100 86 92 85 65 80
c
MI
Revrplate
%S
26 " 4
100
20 " 1 21 " 5 23 " 3 25 " 4 23 " 4 y
98 98 91 77 83
19 " 3
100
25 " 1 29 " 7 29 " 2 33 " 5 37 " 4 "
76 86 96 73 75
19 " 2 18 " 4 19 " 3 19 " 3 17 " 2 17 " 1 y
100 89 64 96 70 94
22 " 1
100
1.1
25 " 4
85
1.2
27 " 5
83
1.0 1.0 1.0
21 " 5 22 " 4 23 " 5 y
72 50 67
21 " 3 21 " 4 20 " 3 17 " 2 23 " 2 21 " 3 y
100 99 88 96 81 78
0.8 0.8 0.9 1.0 0.9
19 " 3 21 " 1 20 " 4 22 " 5 19 " 3 18 " 1
100 90 80 75 50 31
1.3 1.5 1.5 1.7 1.9
y
Response NC Harmaline
TA98 qS9 Revrplate
y
Response NC Harmol
a
17 " 2 18 " 4 19 " 4 17 " 5 18 " 3 16 " 2 y
100 96 88 80 89 67
20 " 5 18 " 4 21 " 4 19 " 6 20 " 4 20 " 5
100 105 95 69 69 86
0.9 1.0 1.0 0.9 0.9
y
100 200 300 400 500
1.2 1.0 1.1 1.4 1.0
16 " 2 20 " 1 16 " 3 18 " 4 23 " 8 16 " 5 y
100 101 61 74 88 97
1.0 1.0 0.8 1.1 1.0
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
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Table 4 Induction of Hisq revertants in S. typhimurium TA97 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance
Dose
TA97 yS9
Žmgrplate.
MI
10 20 30 40 50 60 80 100
1.0 1.2 1.9 2.1 1.9
d
NC Harman
Response NC Harmine
e
10 20 30 40 50 100 150 200 250
0.9 0.9 0.9 0.9 0.8
10 20 30 40 50 60 80 100
1.9 2.0 2.0 2.0 1.4
Response
%S 100 103 104 93 91 78
c
MI
Revrplate
%S
116 " 16
100
0.9
105 " 15
103
0.9
100 " 14
79
1.0 1.2 1.1
114 " 9 138 " 16 131 " 8 "
78 78 70
110 " 9 100 " 2 104 " 12 96 " 8 97 " 15 85 " 1
100 77 79 71 62 47
86 " 14
100
109 " 20 129 " 14 141 " 15 115 " 15 126 " 7 "
101 59 39 31 11
88 " 9
100
1.1
98 " 2
96
1.0
91 " 8
83
1.1 1.1 1.2
98 " 2 95 " 9 102 " 11 y
99 99 98
110 " 12
100
101 " 2 108 " 7 110 " 21 108 " 7 101 " 8 y
86 72 66 52 56
110 " 6 103 " 13 95 " 2 107 " 17 110 " 10 116 " 16 y
100 89 94 74 87 75
1.3 1.5 1.6 1.3 1.5
161 " 33 301 " 12 325 " 12 317 " 36 319 " 23 233 " 19
100 74 68 62 78 79
q
10 20 30 40 50 100 150 200 250
1.1 1.1 1.0 1.1 0.9
Response NC Harmalol
108 " 16 109 " 16 130 " 18 207 " 31 228 " 84 204 " 20
b
y
Response NC Harmaline
TA97 qS9 Revrplate
q
Response NC Harmol
a
142 " 16 150 " 19 156 " 24 142 " 25 150 " 20 131 " 12
100 82 87 99 81 80
0.9 1.0 1.0 1.0 0.9
y
100 200 300 400 500
1.1 1.0 0.8 0.7 0.7
141 " 13 152 " 12 140 " 14 106 " 4 104 " 21 103 " 9 y
100 98 93 62 75 87
0.9 0.9 1.0 1.0 1.1
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
The SOS induction factor Ž I . in treated cells was obtained from the rate of b-galactosidase and alkaline-phosphatase activities compared to untreated cells. The result was considered positive when the increase of the induction factor was more than 2.0 combined with an increase in the b-galactosidase activity. 2.5. Salmonellar microsome assay Mutagenicity was measured in the preincubation procedure w20x, using various concentrations of alkaloids with strains TA98, TA97, TA100, and TA102 of Salmonella typhimurium with or without S9 mix. The mixture consisting of the alkaloid to be tested, 500 ml of S9 mix Žin test with metabolization. and 100 ml of the bacterial suspension Ž1–2 = 10 9 cellsrml., was preincubated for 20 min at 378C without shaking. Then, 2000 ml of top agar Ž0.55% agar, 0.55% NaCl, 50 mM L-histidine, 50 mM biotin, pH 7.4, 458C. was added in the test tube and poured onto a Petri dish with minimal agar Ž1.5% agar, Vogel–Bonner E medium, containing 2% glucose.. All assays were carried out in triplicate. After incubation for 48 h, colonies ŽHisq revertants. were counted and the results were expressed as mutagenic index ŽMI s no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control.. For determination of the cytotoxic effects, after preincubation, the mixture as indicated above was diluted in phosphate buffer Ž0.02 M, pH 7.4. to obtain a suspension containing 1–2 = 10 3 cellsrml. A suitable aliquot of this suspension was plated on nutrient agar Ž0.8% bacto nutrient broth ŽDifco., 0.5% NaCl and 1.5% agar. in triplicate. Plates were incubated at 378C for 48 h before counting the colonies. Negative Žappropriate solvent. and positive Ž5 mg sodium azide per plate for TA100 strain; 0.5 mg 4-nitroquinoline 1-oxide Ž4-NQO. per plate for TA98,
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TA97, and TA102 strains. controls were included in each assay. Aflatoxin B1 Ž1 mg per plate. was used as positive control for the metabolic activation for all strains. The compound was considered positive for mutagenicity when: Ža. the number of revertants was at least double the spontaneous yield ŽMI G 2.; Žb. a significant response for analysis of variance Ž p F 0.05. was found; and Žc. a reproducible positive dose–response curve Ž p F 0.01. was present, evaluated by the Salmonel software w27x. When only two of these criteria were met, the compound was considered to present signs of positivity. This evaluation followed the criteria used by Vargas et al. w28,29x. The sample was classified as cytotoxic when cell survival was less than 70% in the cell viability test. 2.6. Animals Young male and female Swiss Webster mice weighing approximately 25 g Ž5–7 weeks of age. at the time of treatment, were obtained from Laboratorio ´ Regional de Apoio Animal Porto Alegre, RS, Brazil, and employed in the micronucleus assay. Food and water were available ad libitum. All mice were acclimatized for at least one week prior to dosing. Ten animals Ž5 male and 5 female. were randomly allocated to the different groups ŽTables 7 and 8.. 2.7. Micronucleus assay The micronucleus assay was performed according to the US Environmental Protection Agency Gene Tox Program w24x. The test groups were dosed with a single intraperitoneal maximum tolerated dose ŽMTD. established by a preliminary test. Two test groups were used, killed 24 and 48 h after dosing Ž30 mgrkg of harman in dimethylsulfoxide and water 1:1, and 46 mgrkg of harmine in water., parallel to a positive control group Ždosed with 40 mgrkg of
Notes to Table 4: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 459 " 41; ŽqS9. aflatoxin B1 Ž1 mgrplate., 309 " 45. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve p F 0.01; MI - 2..
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Table 5 Induction of Hisq revertants in S. typhimurium TA100 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance NC d Harman
Response NC Harmine
Dose
TA100 yS9
Žmgrplate.
MI
10 20 30 40 50 60 80 100
1.1 1.1 1.1 1.1 1.1
e
Response a
%S 100 104 100 87 77 61
c
MI
Revrplate
%S
105 " 10
100
1.0
108 " 3
90
1.2
123 " 11
90
1.0 1.1 1.1
110 " 5 112 " 8 117 " 16 y
84 88 92
1.1 1.0 1.1 0.9 0.9
114 " 9 123 " 10 114 " 6 122 " 7 103 " 7 101 " 5 y
100 90 70 56 54 50
1.0 1.0 1.0 1.1 1.0
139 " 5 142 " 3 139 " 4 145 " 6 147 " 12 146 " 9 y
100 106 76 76 80 80
10 20 30 40 50
1.1 1.1 0.9 0.9 0.9
111 " 7 119 " 9 118 " 5 104 " 19 103 " 3 95 " 1 y
100 94 91 77 59 53
1.1 1.2 1.1 1.1 0.9
111 " 12 121 " 14 130 " 15 121 " 10 116 " 4 97 " 9 y
100 67 87 88 90 97
50 100 150 200 250
1.0 1.0 1.0 1.0 1.0
114 " 6 115 " 5 119 " 15 114 " 14 115 " 9 111 " 4 y
100 92 100 69 56 43
1.1 1.1 1.0 1.0 1.1
106 " 9 112 " 12 112 " 11 105 " 18 105 " 10 117 " 12 y
100 93 98 96 79 78
50 100 150 200 250
1.1 1.1 1.2 1.1 1.0
153 " 27 164 " 2 168 " 22 176 " 10 161 " 14 159 " 15 y
100 90 73 57 65 59
1.0 1.0 1.1 1.0 1.0
112 " 11 116 " 12 115 " 15 121 " 19 113 " 11 114 " 11 y
100 54 73 92 86 80
Response NC Harmalol
110 " 10 121 " 25 117 " 7 123 " 17 121 " 9 126 " 1
b
10 20 30 40 50
Response NC Harmaline
TA100 qS9 Revrplate
y
Response NC Harmol
a
Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. sodium azide Ž5 mgrplate., 1366 " 230; ŽqS9. aflatoxin B1 Ž1 mgrplate., 435 " 84. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2.. b
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
cyclophosphamide. and two negative control groups one dosed with water and the other with DMSOrwater 1:1. Mice were killed by cervical dislocation for bone marrow analysis. Bone marrow from both femurs was suspended in fetal bovine serum and smears on clean glass slides were made according to MacGregor et al. w30x. Slides were air-dried, fixed in methanol, stained by the May– GrunwaldrGiemsa combination and coded. The proportion of polychromatic erythrocytes ŽPCE. was evaluated in 1000 erythrocytes and the frequency of micronuclei in 2000 PCE for each animal. The x 2test was used for the comparison between test group and negative control. Doubling of the negative control frequency or demonstration of a statistically significant difference Ž p F 0.05. between micronucleus frequencies in treated and control animals was considered to be sufficient for a positive result w24x. 3. Results 3.1. Induction of SOS response Amongst the alkaloids tested only harman Ž10 mgrtest. induced the SOS response, in the absence of metabolic activation, by a factor of 2.41 ŽTable 1.. At high doses Žup to 10 mgrtest., harman, harmine, and harmaline inhibited the constitutive enzyme alkaline phosphatase, which indicates a toxic effect of the treatments. Thus, the observed increase in the enzymatic ratio may be due to the loss of alkaline phosphatase activity and not to true SOS induction ŽTable 1.. In the presence of the metabolic activation mixture the cytotoxic effect was observed only with harmaline ŽTable 2.. The high level of induction for harmalol ŽTables 1 and 2. was considered as falsepositive due to interference of absorption by this alkaloid in the spectrometric measurement of the enzymes. 3.2. Salmonella mutagenicity assay The cytotoxicity, calculated from cell survival, decreased in the following order: harmine) harman ) harmol) Žharmaline, harmalol.. The cytotoxic effect was inhibited by the addition of S9 mix, raising the cytotoxic concentration range for harmine, harman, harmol, harmaline up to 5-fold ŽTables 3–6..
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As observed in Table 3, the aromatic b-carbolinic alkaloids, harman and harmol, were not able to induce frameshift mutation in the TA98 strain. However, some of these alkaloids showed positive results in TA97 in the absence of metabolic activation ŽTable 4.. This strain has a hot spot of G–C pairs in the hisD6610 mutation that make it highly sensitive to frameshift mutagens w31x. In the presence of metabolization, signs of positivity were found only for harman while harmol was not mutagenic ŽTable 4.. At maximum soluble concentrations, harmine showed signs of positivity to both frameshift strains in the presence of metabolic activation, whereas the toxicity of this alkaloid decreased ŽTables 3 and 4.. When harman and harmine were tested in TA102, which detects oxidative and alkylating mutagens and active forms of oxygen w32x, signs of positivity were observed with and without microsomal activation ŽTable 6.. All aromatic b-carbolinic alkaloids tested did not show mutagenic activity in the base-substitution strain TA100 with as well as without metabolic activation ŽTable 5.. Alkaloids of the dihydro-b-carbolinic group, harmaline and harmalol, were not mutagenic in TA98, TA97, and TA100 strains neither in presence nor in absence of metabolic activation ŽTables 3–5.. Only harmaline showed signs of positivity for TA102 in the presence of S9 mix ŽTable 6.. 3.3. Induction of micronucleus response Micronucleus assay results are presented in Tables 7 and 8, which show the mean and standard deviation of frequency of PCE and micronucleated polychromatic erythrocytes ŽMNPCE., individually and by sex and groups. At the doses used, no toxicity in bone marrow was detected. The test groups were statistically similar to their respective negative control, when compared by the x 2-test. Based on these results, harman and harmine are probably not able to induce chromosome mutations in vivo.
4. Discussion Table 9 summarizes the results on the genotoxicity of the b-carboline alkaloids. The positive results to mutagenicity ŽTA97-S9. for harman and harmol
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
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Table 6 Induction of Hisq revertants in S. typhimurium TA102 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Dose mgrplate
Substance
TA102 yS9 MI
NC d Harman
Response NC Harmine
10 20 30 40 50 60 80 100
0.9 1.1 1.1 1.2 1.3
e
5 10 15 20 25 40 60 80 100
1.1 1.2 1.4 1.6 1.6
10 20 30 40 50 60 80 100
1.0 1.0 1.0 1.0 0.9
Response
%S 100 98 97 70 67 69
c
MI
Revrplate
%S
305 " 3
100
1.1
320 " 4
99
1.1
342 " 6
99
1.2 1.3 1.2
371 " 4 384 " 28 376 " 8 "
92 90 76
238 " 16 250 " 29 297 " 11 323 " 5 375 " 14 391 " 9
100 89 81 71 72 41
380 " 29
100
1.2
469 " 27
103
1.3 1.5 1.3 1.5
486 " 34 552 " 5 496 " 45 588 " 23 "
81 73 65 49
323 " 25 337 " 35 337 " 17 330 " 13 311 " 4 297 " 10
100 95 83 77 80 59
275 " 21
100
1.0
285 " 18
84
1.1
293 " 23
85
1.1 1.1 1.0
315 " 17 298 " 28 279 " 3 y
86 81 85
290 " 18
100
1.1
306 " 28
84
1.0
295 " 21
59
1.0 1.1 1.3
300 " 19 321 " 23 380 " 23 "
61 59 38
360 " 18 387 " 18 392 " 23 395 " 33 407 " 26 412 " 18 y
100 72 80 96 86 63
y
50 100 150 200 250 300 400 500
1.0 1.0 1.0 0.9 0.8
Response NC Harmalol
280 " 15 251 " 35 304 " 11 321 " 17 325 " 15 356 " 16
b
"
Response NC Harmaline
TA102 qS9 Revrplate
"
Response NC Harmol
a
313 " 23 325 " 23 325 " 11 309 " 27 268 " 17 260 " 13
100 122 78 87 61 61
y
50 100 150 200 250
1.0 1.0 1.0 1.0 0.8
244 " 3 239 " 9 243 " 15 246 " 6 241 " 32 205 " 13 y
100 96 92 75 67 71
1.1 1.1 1.1 1.1 1.1
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
145
Table 7 Results of the micronucleus assay after a single treatment of male ŽM. and female ŽF. Swiss Webster mice with harman Group
a
Sex
Frequencies of polychromatic erythrocytes ŽPCE. in 1000 erythrocytes Mean " SD
Individual results Cq Cy T1 T2
M F M F M F M F
528 476 498 467 516 544 567 534
453 500 533 446 549 550 570 565
436 513 487 511 561 508 568 440
470 443 534 488 540 544 528 477
510 601 459 493 508 538 486 449
by sex
by group
479" 39 507" 59 502" 32 481" 25 535" 22 537" 17 544" 37 493" 54
493 " 49 492 " 29 536 " 19 518 " 51
Frequencies of micronucleated polychromatic erythrocytes ŽMNPCE. in 2000 PCE Mean " SD
Individual results Cq Cy T1 T2
M F M F M F M F
66 51 7 7 9 9 8 8
47 51 7 7 8 8 8 7
44 52 6 6 7 7 9 7
65 50 7 7 8 7 7 7
57 45 6 7 10 7 7 7
by sex
by group
55.8 " 1.0 49.8 " 2.8 6.6 " 0.5 6.8 " 0.4 8.4 " 1.1 7.6 " 0.9 7.8 " 0.8 7.2 " 0.4
52.8 " 7.7 6.7 " 0.5 8.0 " 1.1 7.5 " 0.7
a
Cq, positive control group; Cy, negative control group; T1 , test group killed 24 h after treatment; T2 , test group killed 48 h after treatment.
can be considered very weak frameshift mutation responses, with just over a two-fold increase in revertants over background observed in each case ŽTable 4.. In the SOS chromotest, only one positive result was found; harman weakly induced SOS functions without S9 mix, while the other related alkaloids showed non-induction or false-induction SOS Žsee Tables 1 and 2.. Eight signs of positivity results ŽTable 9. were found in TA102 as well as in frameshift strains TA98 and TA97, indicating marginal mutagenic activities in those cases. The negative results in TA100 indicated that these chemicals cannot be mutagens by base-pair substitution. Furthermore, the chemicals tested in the micronu-
cleus assay, harman and harmine, did not induce micronuclei in bone marrow PCE, suggesting that they cannot cause in vivo mutagenicity. We observed that the cytotoxicity of the alkaloids was reduced in the testing strains due to the use of S9 mix in the Salmonellarmicrosome assay and in the SOS chromotest as well. Harman, for instance, may be tested even at 250 mgrplate in the presence of S9 mix ŽTable 3. while in the absence of metabolization the largest dose was 50 mgrplate. Harmine at the concentrations of 10 and 100 mgrtest without S9 mix was cytotoxic in the SOS chromotest, which can be verified by the slope of UPase ŽTable 1., but this effect was not observed in the presence of
Notes to Table 6: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 2477 " 180; ŽqS9. aflatoxin B1 Ž1 mgrplate., 1013 " 272. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2..
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
146
Table 8 Results of the micronucleus assay after single treatment of male ŽM. and female ŽF. Swiss Webster mice with harmine Group
a
Sex
Frequencies of polychromatic erythrocytes ŽPCE. in 1000 erythrocytes Mean " SD
Individual results Cq
M F M F M F M F
Cy T1 T2
528 476 463 512 545 503 272 515
453 500 434 492 458 451 436 535
436 513 471 519 560 585 426 323
470 443 450 600 440 421 418 395
510 601 357 465 548 585 427 580
by sex
by group
479" 39 507" 59 435" 46 518" 51 510" 57 509" 75 396" 70 470" 107
493 " 49 476 " 63 510 " 63 433 " 94
Frequencies of micronucleated polychromatic erythrocytes ŽMNPCE. in 2000 PCE Mean " SD
Individual results Cq
M F M F M F M F
Cy T1 T2
66 51 6 6 11 7 6 7
47 51 7 6 7 7 7 7
44 52 7 5 7 9 7 6
65 50 6 6 5 7 7 7
57 45 6 7 8 7 7 9
by sex
by group
55.8 " 1.0 49.8 " 2.8 6.4 " 0.5 6.0 " 0.7 7.6 " 2.2 7.4 " 0.9 6.8 " 0.4 7.2 " 1.1
52.8 " 7.7 6.2 " 0.6 7.5 " 1.6 7.0 " 0.8
a
Cq, positive control group; Cy, negative control group; T1 , test group killed 24 h after treatment; T2 , test group killed 48 h after treatment.
metabolization and at the same concentrations ŽTable 2.. The same phenomenon occurred in the test with harmaline at 100 mgrtest ŽTables 1 and 2.. These effects of S9 mix can be explained by a partial detoxification of the alkaloids by reaction with microsomal enzymes of the S9 mix, such as epoxide
hydrolase and cytochrome P-450 mixed function oxygenase system, so that higher doses can be applied w33x. The recent review by Quillardet and Hofnung w23x determined that the correspondence between the SOS chromotest and Salmonellarmicrosome assay results
Table 9 Summary of results on the genotoxicity of the b-carboline alkaloids Alkaloid
Results
a
Mutagenicity TA98
Harman Harmine Harmol Harmaline Harmalol a
SOS induction TA97
TA100
TA102
MN induction
PQ37
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
y y y y y
y " y y y
q y q y y
" " y y y
y y y y y
y y y y y
" " y y y
" " y " y
q y y y y
y y y y y
y y NT NT NT
MN, micronuclei; yS9, without metabolic activation; qS9, with metabolic activation; q, positive; y, negative; ", signs of positivity; NT, not tested.
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
is very high Žover 82%.. Indeed, the induction of b-galactosidase synthesis in the SOS chromotest reflects the level of expression of SOS functions involved in cell division inhibition Ž sulA
147
ing harmol w36,38,39x. In vivo, harmol takes part in reactions of conjugation with glycuronic acid andror sulfate, but in vitro these reactions are poorly operative resulting in the free form w36,40x. Since harmol demonstrated mutagenic activity for TA97 in the direct test ŽTable 4., we may conclude that the mutagenicity obtained in the harmine tests on TA97 with S9 mix may be related to the formation of free harmol in situ. On the other hand, in the micronucleus assay, the putatively formed harmol in harmine-treated mice may have taken part in conjugating reactions resulting in reduced toxicity and lowered effects on the induction of micronuclei. In recent studies w11x, harman, harmine, harmaline, and norharman have been shown to be non-clastogenic in vitro for Chinese hamster ovary cells ŽCHO. in the presence and in the absence of S9 mix. Our results obtained from the micronucleus assay ŽTables 7 and 8. thus confirm in vivo that harman and harmine are not clastogenic as the MN frequency is not significantly increased in mouse PCE. Harman and harmine showed signs of positivity in TA102 with and without metabolic activation, whereas harmaline had the same type of response only in the presence of S9 mix ŽTable 6.. 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 alkylating agents, and has an intact excision–repair pathway w32x. Thus, these signs of positivity in TA102 can be due to DNA adduct formations by the alkaloids or by the metabolites generated by the S9 mix action in the A–T sites of bacterial DNA. Indeed, when harman was administered in the diet of mice, DNA adducts were found in liver and kidney w15x. As reported elsewhere w16,17x, aromatic b-carbolines, such as harman and harmine ŽFig. 1A., interact with DNA by intercalation. The same does not hold for the dihydro-b-carbolines, such as harmaline ŽFig. 1B.. Apparently this is due to the non-planarity of the ring system and the lack of full aromaticity of these dihydro-b-carbolines w17,41,42x. Therefore the activities reported here could be associated with the different abilities of these chemicals to interact with DNA. In fact, we showed that in the bacterial tests, the aromatic b-carbolines were more cytotoxic than the dihydro-b-carbolines. In addition to that, harman, harmine, and harmol, which have a totally aromatic
148
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
structure ŽFig. 1A., showed a weak genotoxic activity or just signs of positivity, while harmaline and harmalol ŽFig. 1B. behave practically as non-genotoxic ŽTable 9.. Interestingly enough, these responses can be related to the degree of aromaticity and planarity of these compounds, inasmuch as observed in the studies on their ability to interact with DNA. It is interesting to note that in the micronucleus assay ŽTables 7 and 8., harman and harmine, which had shown to be the most active alkaloids in the bacterial tests, showed results indicating that they do not induce in vivo mutagenicity. The present results may suggest that these alkaloid b-carbolines probably do not carry long-term genetic risk, although further support is necessary to decide whether those chemicals can be hazardous to human health. Their biological effect still cannot be fully understood, such as that related to their comutagenic activity w8–12x. Furthermore, humans are sufficiently exposed to some of those chemicals, which are both present in plants used for the preparation of hallucinogenic material and medicinal drugs, and in tobacco smoke and well-cooked food w1–5x. As a consequence of these observations, further studies on the genetic toxicology of b-carbolines in other test systems would be worthwhile.
Acknowledgements We thank Drs. Martin Brendel, Tatiana Golubkova and Nikita Khromov-Borisov for helpful reading and discussion of the paper. This work was supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Cientıfico e ´ ŽCNPq., Fundac¸ao Tecnologico ´ ˜ de Amparo a` Pesquisa do Estado do Rio Grande do Sul ŽFAPERGS., and Laboratorio de Genotoxicidade ´ ŽGENOTOX.rCentro de BiotecnologiarUFRGS.
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w30x J.T. MacGregor, J.A. Heddle, M. Hite, B.H. Margolin, C. Ramel, M.F. Salamone, R.R. Tice, D. Wild, Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes, Mutation Res. 189 Ž1987. 103–112. w31x D.E. Levin, E. Yamasaki, B.N. Ames, A new Salmonella tester strain, TA97, for detection of frameshift mutagens, Mutation Res. 94 Ž1982. 315–330. w32x D.E. Levin, M. Hollstein, M.F. Christman, E.A. Schwiers, B.N. Ames, A new Salmonella tester strain ŽTA102. with A–T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. USA 79 Ž1982. 7445–7449. w33x C. de Meester, Genotoxic potential of b-carbolines: a review, Mutation Res. 339 Ž1995. 139–153. w34x G.C. Walker, Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev. 48 Ž1984. 60–93. w35x D.J. Tweedie, M.D. Burke, Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism of the b-carbolines harmine and harmol, Biochem. Pharmacol. 32 Ž1983. 653–663. w36x D.J. Tweedie, M.D. Burke, Metabolism of the b-carbolines, harmine and harmol, by liver microsomes from phenobarbitone or 3-methylcholanthrene-treated mice: identification and quantitation of two novel harmine metabolites, Drug Metab. Dispos. 15 Ž1986. 74–82. w37x Y. Oda, S.-I. Nakamura, I. Oki, Harman and norharman induce SOS responses and frameshift mutations in bacteria, Mutation Res. 208 Ž1988. 39–44. w38x T.A. Slotkin, V. Distefano, Urinary metabolites of harmine in the rat and their inhibition of monoamine oxidase, Biochem. Pharmacol. 19 Ž1970. 125–131. w39x T.A. Slotkin, V. Distefano, A model of harmine metabolism in the rat, J. Pharmacol. Exp. Ther. 174 Ž1970. 456–462. w40x G.J. Mulder, B. Bleeker, UDP glucuronyltransferase and phenol-sulfotransferase from rat liver in vivo and in vitro VI: Species differences in harmol conjugation and elimination in bile and urine in vivo, Biochem. Pharmacol. 24 Ž1975. 1481–1484. w41x G. Duportail, Linear and circular dichroism of harmine and harmaline interacting with DNA, Int. J. Biol. Macromol. 3 Ž1981. 188–193. w42x J. Hardiman, H.L. Carrel, D.E. Zacharias, J.P. Glusker, The structure of harman, a comutagen, Bioorganic Chem. 15 Ž1987. 213–223.
Genotoxic effects of structurally related b-carboline alkaloids Jaqueline N. Picada a,b, Katia V.C.L. da Silva b, Bernardo Erdtmann c , Amelia ´ T. Henriques a, Joao ˜ A.P. Henriques b,) a
b
Curso de Pos-Graduac ¸ao Farmaceuticas e Departamento de Produc¸ao ´ ˜ em Ciencias ˆ ˆ ˜ de Materia ´ Prima da Faculdade de Farmacia, ´ UFRGS, AÕ. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil Departamento de Biofısica e Centro de Biotecnologia, UFRGS, AÕ. Bento Gonc¸alÕes 9500, predio ´ ´ 43421, Campus do Vale, Caixa Postal 15005, CEP 91501-970, Porto Alegre, RS, Brazil c Departamento de Genetica, UFRGS, AÕ. Bento Gonc¸alÕes, 9500, predio ´ ´ 43323, Campus do Vale, CEP 91501-970, Porto Alegre, RS, Brazil Received 17 January 1997; revised 29 April 1997; accepted 2 May 1997
Abstract b-Carboline alkaloids, found in medicinal plants, tobacco smoke and well-cooked foods, have shown a variety of actions in biological systems related to their interaction with DNA. Therefore, these alkaloids can be considered potentially mutagenic. In this work, the genotoxic, mutagenic, and cytotoxic activities of three aromatic b-carboline alkaloids Žharman, harmine, and harmol. and two dihydro-b-carboline alkaloids Žharmaline and harmalol. were evaluated by means of the Salmonellarmicrosome assay Ž Salmonella typhimurium TA98, TA97, TA100, and TA102. and SOS chromotest Ž Escherichia coli PQ37. with and without metabolic activation. Moreover, harman and harmine were analyzed by the micronucleus assay in vivo. It was shown that genotoxicity was inhibited by the addition of S9 mix for aromatic b-carbolines harman and harmol in TA97. However, harmine showed signs of mutagenicity only in the presence of S9 mix in TA98 and TA97 frameshift strains. In the SOS chromotest, only harman induced SOS functions in the absence of S9 mix. Dihydro-b-carbolines were not genotoxic in any of the microorganisms used. The negative responses obtained in the micronucleus assay indicated that harman and harmine were not able to induce chromosomal mutations. q 1997 Elsevier Science B.V. Keywords: b-Carboline; Genotoxicity; Micronucleus assay; Mutagenicity; Salmonellarmicrosome assay; SOS chromotest
1. Introduction b-Carboline alkaloids, also known as harmala’s alkaloids because they were first isolated from Peganum harmala ŽZygophillaceae., are natural products that have been consumed as hallucinogenic drinks, snuffs and other forms in the Amazon basin, ) Corresponding author. Fax: q55 Ž51. 336-2779; e-mail: [email protected]
where they are found in Banisteriopsis spp. and Tetrapteris spp. ŽMalpighiaceae., Virola spp. ŽMyristicaceae. and Anadenanthera spp. ŽLeguminosae. w1,2x. Passiflora spp. ŽPassifloraceae. Žpopularly know as Maracujas ´ ., which contains b-carboline alkaloids, such as harman, is one of the plants widely used in medical practice in Brazil, mainly for its anti-spasmodic and sedative properties w1x. In addition to their natural occurrence in plants, bcarbolines have been found in a number of other
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 1 6 - 4
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J.N. Picada et al.r Mutation Research 379 (1997) 135–149
sources. Harman and norharman have also been detected in tobacco smoke and well-cooked foods, reflecting their formation from tryptophan by pyrolysis w3–5x. These compounds have been of interest for some time because of the variety of actions which they evoke in biological systems w1,6,7x. Harman and norharman induce a co-mutagenic effect with a number of compounds in Salmonella typhimurium TA98 and TA100 w8–10x and in mammalian cell culture w11,12x. Both are cytotoxic and mutagenic to Chinese hamster lung cells ŽCHL cells. w13x. They are also reported to induce sister chromatid exchange ŽSCE. in human peripheral lymphocytes w14x. Treatment of mice with 0.1% harman in their diet for 4 weeks resulted in DNA adducts in the liver and kidney w15x. Their activities in prokaryotic and eukaryotic cells have been attributed in part to the ability of these chemicals to interact with DNA. Hayashi et al. w16x found that harman and norharman react with DNA by intercalation: DNA induced a quenching of the fluorescence of norharman, and a marked red shift and hypochromism of the absorption spectra of norharman and harman. Circular supercoiled DNA is unwound by norharman. Hayashi et al. w16x reported that harman intercalates more easily into DNA than norharman. Duportail and Lami w17x showed that the hydrogenation of a double bond of the pyridine ring converting harmine, an aromatic b-carboline, to harmaline, a dihydro-b-carboline, greatly alters the interaction of the molecule with DNA. Measurements of the viscosity indicated intercalation of harmine only. It is very well known that the mutagenic and carcinogenic effects of the various compounds are related to their intercalation with DNA w18,19x. So, it is very important to find out if b-carboline alkaloids show genotoxic and mutagenic activities in prokaryotic cells. Since these compounds are present in human food and induce mutagenesis in mammalian cells in culture, it is also necessary to study their mutagenicity in vivo. Thus, the genotoxic activity of three aromatic b-carboline alkaloids, harman, harmine, and harmol, and two dihydro-b-carboline alkaloids, harmaline and harmalol was evaluated by means of the Salmonellarmicrosome assay w20,21x and the SOS chromotest w22,23x. Moreover, harman and harmine were tested for their ability to induce micronuclei in mice bone marrow polychromatic erythrocytes w24,25x.
2. Materials and methods 2.1. Chemical products The structural formula and the chemical name of each b-carboline alkaloid tested for genotoxicity is shown in Fig. 1. The aromatic b-carbolines, harman ŽCAS 2165584-5; not specified purity., harmine ŽCAS 343-27-1; approx. 98% purity., and harmol ŽCAS 149022-16-2; not specified purity. hydrochlorides, and the dihydro-b-carbolines, harmaline ŽCAS 6027-98-1; approx. 95% purity. and harmalol ŽCAS 6028-07-5; approx. 90% purity. hydrochloride, were obtained from Sigma Co ŽSt. Louis, MO, USA.. To carry out the SOS chromotest, Salmonellarmicrosome assay, and micronucleus assay, harmine was dissolved in sterile distilled water while the other four alkaloids tested were dissolved in spectrophotometric-grade dimethylsulfoxide ŽDMSO. and sterile distilled water Ž1:1.. 2.2. Strains Escherichia coli SOS-chromotest tester strain PQ37, which has the following genotype: Fy, thr leu his-4 pyrD thi galE galk (galT) lac169 srl300
Fig. 1. Chemical structure of the b-carboline alkaloids. A: aromatic b-carbolines. B: dihydro-b-carbolines.
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
tut Pasteur, Paris ŽFrance.. The Salmonella typhimurium strains TA98, TA97, TA100, and TA102, as described by Maron and Ames w20x, were kindly provided by Dr. B.N. Ames, University of California, Berkeley, CA ŽUSA..
137
ŽMoltox e .. The S9 metabolic activation mixture was prepared according to Quillardet and Hofnung w22x for the SOS chromotest and according to Maron and Ames w20x for the Salmonellarmicrosome assay. 2.4. SOS chromotest
2.3. Microsomal fraction The postmicrosomal fraction S9 was prepared from livers of Sprague–Dawley rats pretreated with polychlorinated biphenyl mixture ŽAroclor 1254., purchased from Molecular Toxicology Inc.
The SOS chromotest was performed according to Quillardet and Hofnung w22x. An exponential-phase culture of Escherichia coli PQ37, grown in LB medium Ž1% bacto tryptone ŽDifco., 0.5% bacto yeast extract ŽDifco. and 1% NaCl. plus ampicillin
Table 1 SOS function inducing activity of b-carboline alkaloids in E. coli PQ37 in the SOS chromotest without metabolic activation Substance 4-NQO
a
Harman
Harmine
Harmol
Harmaline
Harmalol
a
Dose Žmgrtest.
b-Galactosidase ŽB. Žunits.
Alkaline phosphatase ŽP. Žunits.
BrP
Induction factor
0 0.01 0.05 0 0.1 1 5 10 50 0 0.1 1 10 100 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 500 0 0.01 0.1 1 10
410 1225 1605 46 83 80 84 95 191 46 69 69 153 46 521 577 569 569 648 267 410 561 474 421 456 596 900 521 584 548 783 1170
1380 1670 1410 582 571 571 607 500 430 582 554 582 357 143 1530 1530 1400 1330 1130 450 1380 1740 1550 1690 1460 921 320 1530 1530 1500 1450 1600
0.297 0.733 1.138 0.079 0.145 0.140 0.138 0.190 0.444 0.079 0.125 0.119 0.429 0.322 0.341 0.377 0.406 0.428 0.573 0.593 0.297 0.322 0.306 0.249 0.312 0.647 2.813 0.341 0.382 0.365 0.540 0.731
1.00 2.47 3.83 1.00 1.84 1.77 1.75 2.41 5.62 1.00 1.58 1.51 5.43 4.08 1.00 1.11 1.19 1.26 1.68 1.74 1.00 1.08 1.03 0.84 1.05 2.18 9.47 1.00 1.12 1.07 1.58 2.14
b
Positive control. The induction factor at concentration C is defined as Ic s R crR 0 , where R c and R 0 are the ratios of b-galactosidase and alkaline phosphatase activities with and without added DNA-damaging agent. b
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
138
Table 2 SOS function inducing activity of b-carboline alkaloids in E. coli PQ37 in the SOS chromotest with metabolic activation Substance AFB1
a
Harman
Harmine
Harmol
Harmaline
Harmalol
Dose Žmgrtest.
b-Galactosidase ŽB. Žunits.
Alkaline phosphatase ŽP. Žunits.
BrP
Induction factor
0 0.005 0.02 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 250 0 0.1 1 10
410 1230 1650 439 490 488 488 455 439 488 520 520 390 411 457 457 411 381 439 455 423 553 683 878 411 488 457 854
1380 1270 827 976 1105 1140 1100 793 976 1100 1070 1590 1100 1360 1300 1220 1330 1430 976 945 1070 915 854 793 1360 1250 1270 1300
0.297 0.969 1.995 0.450 0.443 0.428 0.444 0.574 0.450 0.444 0.486 0.327 0.355 0.302 0.352 0.375 0.309 0.266 0.450 0.481 0.395 0.604 0.800 1.107 0.302 0.390 0.360 0.657
1.00 3.25 6.69 1.00 0.99 0.95 0.99 1.28 1.00 0.99 1.08 0.73 0.79 1.00 1.17 1.24 1.02 0.88 1.00 1.07 0.88 1.34 1.78 2.46 1.00 1.29 1.19 2.18
b
a
Positive control. The induction factor at concentration C is defined as Ic s R crR 0 , where R c and R 0 are the ratios of b-galactosidase and alkaline phosphatase activities with and without added DNA-damaging agent. b
Ž20 mgrml. at 378C, was diluted 1:10 into fresh medium or in S9 mix for metabolic activation. Aliquots of 600 ml were distributed into glass test tubes containing 20 ml of the alkaloids to be tested. As positive controls, aflatoxin B1 ŽAFB1. and 4nitroquinoline 1-oxide Ž4-NQO. Žboth from Sigma Chemical, St. Louis, MO, USA. were used with and without metabolization. After 2 h of incubation at
378C with shaking, 300 ml samples were taken to assay for b-galactosidase and alkaline-phosphatase activities. In this assay, the sfiA gene linked bgalactosidase activity is determined as a measure of the induction of the SOS repair system. The activity of the constitutive enzyme alkaline phosphatase was used as a measure of protein synthesis and toxicity. Enzyme activities were assayed in Miller units w26x.
Notes to Table 3: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values of at least two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 194 " 25; ŽqS9. aflatoxin B1 Ž1 mgrplate., 476 " 41. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2..
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
139
Table 3 Induction of Hisq revertants in S. typhimurium TA98 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance NC d Harman
Response NC Harmine
Dose
TA98 yS9
Žmgrplate.
MI
10 20 30 40 50 100 150 200 250
1.1 1.0 1.0 0.9 0.9
e
10 20 30 40 50 100 150 200 250
1.1 1.1 1.1 1.0 0.9
20 40 60 80 100
1.0 1.1 1.0 1.1 0.9
50 100 150 200 250 300 400 500
0.9 1.0 1.0 1.0 1.0
Response NC Harmalol
Response
18 " 3 20 " 4 18 " 2 17 " 1 16 " 2 16 " 1
b
%S 100 86 92 85 65 80
c
MI
Revrplate
%S
26 " 4
100
20 " 1 21 " 5 23 " 3 25 " 4 23 " 4 y
98 98 91 77 83
19 " 3
100
25 " 1 29 " 7 29 " 2 33 " 5 37 " 4 "
76 86 96 73 75
19 " 2 18 " 4 19 " 3 19 " 3 17 " 2 17 " 1 y
100 89 64 96 70 94
22 " 1
100
1.1
25 " 4
85
1.2
27 " 5
83
1.0 1.0 1.0
21 " 5 22 " 4 23 " 5 y
72 50 67
21 " 3 21 " 4 20 " 3 17 " 2 23 " 2 21 " 3 y
100 99 88 96 81 78
0.8 0.8 0.9 1.0 0.9
19 " 3 21 " 1 20 " 4 22 " 5 19 " 3 18 " 1
100 90 80 75 50 31
1.3 1.5 1.5 1.7 1.9
y
Response NC Harmaline
TA98 qS9 Revrplate
y
Response NC Harmol
a
17 " 2 18 " 4 19 " 4 17 " 5 18 " 3 16 " 2 y
100 96 88 80 89 67
20 " 5 18 " 4 21 " 4 19 " 6 20 " 4 20 " 5
100 105 95 69 69 86
0.9 1.0 1.0 0.9 0.9
y
100 200 300 400 500
1.2 1.0 1.1 1.4 1.0
16 " 2 20 " 1 16 " 3 18 " 4 23 " 8 16 " 5 y
100 101 61 74 88 97
1.0 1.0 0.8 1.1 1.0
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
140
Table 4 Induction of Hisq revertants in S. typhimurium TA97 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance
Dose
TA97 yS9
Žmgrplate.
MI
10 20 30 40 50 60 80 100
1.0 1.2 1.9 2.1 1.9
d
NC Harman
Response NC Harmine
e
10 20 30 40 50 100 150 200 250
0.9 0.9 0.9 0.9 0.8
10 20 30 40 50 60 80 100
1.9 2.0 2.0 2.0 1.4
Response
%S 100 103 104 93 91 78
c
MI
Revrplate
%S
116 " 16
100
0.9
105 " 15
103
0.9
100 " 14
79
1.0 1.2 1.1
114 " 9 138 " 16 131 " 8 "
78 78 70
110 " 9 100 " 2 104 " 12 96 " 8 97 " 15 85 " 1
100 77 79 71 62 47
86 " 14
100
109 " 20 129 " 14 141 " 15 115 " 15 126 " 7 "
101 59 39 31 11
88 " 9
100
1.1
98 " 2
96
1.0
91 " 8
83
1.1 1.1 1.2
98 " 2 95 " 9 102 " 11 y
99 99 98
110 " 12
100
101 " 2 108 " 7 110 " 21 108 " 7 101 " 8 y
86 72 66 52 56
110 " 6 103 " 13 95 " 2 107 " 17 110 " 10 116 " 16 y
100 89 94 74 87 75
1.3 1.5 1.6 1.3 1.5
161 " 33 301 " 12 325 " 12 317 " 36 319 " 23 233 " 19
100 74 68 62 78 79
q
10 20 30 40 50 100 150 200 250
1.1 1.1 1.0 1.1 0.9
Response NC Harmalol
108 " 16 109 " 16 130 " 18 207 " 31 228 " 84 204 " 20
b
y
Response NC Harmaline
TA97 qS9 Revrplate
q
Response NC Harmol
a
142 " 16 150 " 19 156 " 24 142 " 25 150 " 20 131 " 12
100 82 87 99 81 80
0.9 1.0 1.0 1.0 0.9
y
100 200 300 400 500
1.1 1.0 0.8 0.7 0.7
141 " 13 152 " 12 140 " 14 106 " 4 104 " 21 103 " 9 y
100 98 93 62 75 87
0.9 0.9 1.0 1.0 1.1
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
The SOS induction factor Ž I . in treated cells was obtained from the rate of b-galactosidase and alkaline-phosphatase activities compared to untreated cells. The result was considered positive when the increase of the induction factor was more than 2.0 combined with an increase in the b-galactosidase activity. 2.5. Salmonellar microsome assay Mutagenicity was measured in the preincubation procedure w20x, using various concentrations of alkaloids with strains TA98, TA97, TA100, and TA102 of Salmonella typhimurium with or without S9 mix. The mixture consisting of the alkaloid to be tested, 500 ml of S9 mix Žin test with metabolization. and 100 ml of the bacterial suspension Ž1–2 = 10 9 cellsrml., was preincubated for 20 min at 378C without shaking. Then, 2000 ml of top agar Ž0.55% agar, 0.55% NaCl, 50 mM L-histidine, 50 mM biotin, pH 7.4, 458C. was added in the test tube and poured onto a Petri dish with minimal agar Ž1.5% agar, Vogel–Bonner E medium, containing 2% glucose.. All assays were carried out in triplicate. After incubation for 48 h, colonies ŽHisq revertants. were counted and the results were expressed as mutagenic index ŽMI s no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control.. For determination of the cytotoxic effects, after preincubation, the mixture as indicated above was diluted in phosphate buffer Ž0.02 M, pH 7.4. to obtain a suspension containing 1–2 = 10 3 cellsrml. A suitable aliquot of this suspension was plated on nutrient agar Ž0.8% bacto nutrient broth ŽDifco., 0.5% NaCl and 1.5% agar. in triplicate. Plates were incubated at 378C for 48 h before counting the colonies. Negative Žappropriate solvent. and positive Ž5 mg sodium azide per plate for TA100 strain; 0.5 mg 4-nitroquinoline 1-oxide Ž4-NQO. per plate for TA98,
141
TA97, and TA102 strains. controls were included in each assay. Aflatoxin B1 Ž1 mg per plate. was used as positive control for the metabolic activation for all strains. The compound was considered positive for mutagenicity when: Ža. the number of revertants was at least double the spontaneous yield ŽMI G 2.; Žb. a significant response for analysis of variance Ž p F 0.05. was found; and Žc. a reproducible positive dose–response curve Ž p F 0.01. was present, evaluated by the Salmonel software w27x. When only two of these criteria were met, the compound was considered to present signs of positivity. This evaluation followed the criteria used by Vargas et al. w28,29x. The sample was classified as cytotoxic when cell survival was less than 70% in the cell viability test. 2.6. Animals Young male and female Swiss Webster mice weighing approximately 25 g Ž5–7 weeks of age. at the time of treatment, were obtained from Laboratorio ´ Regional de Apoio Animal Porto Alegre, RS, Brazil, and employed in the micronucleus assay. Food and water were available ad libitum. All mice were acclimatized for at least one week prior to dosing. Ten animals Ž5 male and 5 female. were randomly allocated to the different groups ŽTables 7 and 8.. 2.7. Micronucleus assay The micronucleus assay was performed according to the US Environmental Protection Agency Gene Tox Program w24x. The test groups were dosed with a single intraperitoneal maximum tolerated dose ŽMTD. established by a preliminary test. Two test groups were used, killed 24 and 48 h after dosing Ž30 mgrkg of harman in dimethylsulfoxide and water 1:1, and 46 mgrkg of harmine in water., parallel to a positive control group Ždosed with 40 mgrkg of
Notes to Table 4: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 459 " 41; ŽqS9. aflatoxin B1 Ž1 mgrplate., 309 " 45. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve p F 0.01; MI - 2..
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142
Table 5 Induction of Hisq revertants in S. typhimurium TA100 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Substance NC d Harman
Response NC Harmine
Dose
TA100 yS9
Žmgrplate.
MI
10 20 30 40 50 60 80 100
1.1 1.1 1.1 1.1 1.1
e
Response a
%S 100 104 100 87 77 61
c
MI
Revrplate
%S
105 " 10
100
1.0
108 " 3
90
1.2
123 " 11
90
1.0 1.1 1.1
110 " 5 112 " 8 117 " 16 y
84 88 92
1.1 1.0 1.1 0.9 0.9
114 " 9 123 " 10 114 " 6 122 " 7 103 " 7 101 " 5 y
100 90 70 56 54 50
1.0 1.0 1.0 1.1 1.0
139 " 5 142 " 3 139 " 4 145 " 6 147 " 12 146 " 9 y
100 106 76 76 80 80
10 20 30 40 50
1.1 1.1 0.9 0.9 0.9
111 " 7 119 " 9 118 " 5 104 " 19 103 " 3 95 " 1 y
100 94 91 77 59 53
1.1 1.2 1.1 1.1 0.9
111 " 12 121 " 14 130 " 15 121 " 10 116 " 4 97 " 9 y
100 67 87 88 90 97
50 100 150 200 250
1.0 1.0 1.0 1.0 1.0
114 " 6 115 " 5 119 " 15 114 " 14 115 " 9 111 " 4 y
100 92 100 69 56 43
1.1 1.1 1.0 1.0 1.1
106 " 9 112 " 12 112 " 11 105 " 18 105 " 10 117 " 12 y
100 93 98 96 79 78
50 100 150 200 250
1.1 1.1 1.2 1.1 1.0
153 " 27 164 " 2 168 " 22 176 " 10 161 " 14 159 " 15 y
100 90 73 57 65 59
1.0 1.0 1.1 1.0 1.0
112 " 11 116 " 12 115 " 15 121 " 19 113 " 11 114 " 11 y
100 54 73 92 86 80
Response NC Harmalol
110 " 10 121 " 25 117 " 7 123 " 17 121 " 9 126 " 1
b
10 20 30 40 50
Response NC Harmaline
TA100 qS9 Revrplate
y
Response NC Harmol
a
Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. sodium azide Ž5 mgrplate., 1366 " 230; ŽqS9. aflatoxin B1 Ž1 mgrplate., 435 " 84. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2.. b
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
cyclophosphamide. and two negative control groups one dosed with water and the other with DMSOrwater 1:1. Mice were killed by cervical dislocation for bone marrow analysis. Bone marrow from both femurs was suspended in fetal bovine serum and smears on clean glass slides were made according to MacGregor et al. w30x. Slides were air-dried, fixed in methanol, stained by the May– GrunwaldrGiemsa combination and coded. The proportion of polychromatic erythrocytes ŽPCE. was evaluated in 1000 erythrocytes and the frequency of micronuclei in 2000 PCE for each animal. The x 2test was used for the comparison between test group and negative control. Doubling of the negative control frequency or demonstration of a statistically significant difference Ž p F 0.05. between micronucleus frequencies in treated and control animals was considered to be sufficient for a positive result w24x. 3. Results 3.1. Induction of SOS response Amongst the alkaloids tested only harman Ž10 mgrtest. induced the SOS response, in the absence of metabolic activation, by a factor of 2.41 ŽTable 1.. At high doses Žup to 10 mgrtest., harman, harmine, and harmaline inhibited the constitutive enzyme alkaline phosphatase, which indicates a toxic effect of the treatments. Thus, the observed increase in the enzymatic ratio may be due to the loss of alkaline phosphatase activity and not to true SOS induction ŽTable 1.. In the presence of the metabolic activation mixture the cytotoxic effect was observed only with harmaline ŽTable 2.. The high level of induction for harmalol ŽTables 1 and 2. was considered as falsepositive due to interference of absorption by this alkaloid in the spectrometric measurement of the enzymes. 3.2. Salmonella mutagenicity assay The cytotoxicity, calculated from cell survival, decreased in the following order: harmine) harman ) harmol) Žharmaline, harmalol.. The cytotoxic effect was inhibited by the addition of S9 mix, raising the cytotoxic concentration range for harmine, harman, harmol, harmaline up to 5-fold ŽTables 3–6..
143
As observed in Table 3, the aromatic b-carbolinic alkaloids, harman and harmol, were not able to induce frameshift mutation in the TA98 strain. However, some of these alkaloids showed positive results in TA97 in the absence of metabolic activation ŽTable 4.. This strain has a hot spot of G–C pairs in the hisD6610 mutation that make it highly sensitive to frameshift mutagens w31x. In the presence of metabolization, signs of positivity were found only for harman while harmol was not mutagenic ŽTable 4.. At maximum soluble concentrations, harmine showed signs of positivity to both frameshift strains in the presence of metabolic activation, whereas the toxicity of this alkaloid decreased ŽTables 3 and 4.. When harman and harmine were tested in TA102, which detects oxidative and alkylating mutagens and active forms of oxygen w32x, signs of positivity were observed with and without microsomal activation ŽTable 6.. All aromatic b-carbolinic alkaloids tested did not show mutagenic activity in the base-substitution strain TA100 with as well as without metabolic activation ŽTable 5.. Alkaloids of the dihydro-b-carbolinic group, harmaline and harmalol, were not mutagenic in TA98, TA97, and TA100 strains neither in presence nor in absence of metabolic activation ŽTables 3–5.. Only harmaline showed signs of positivity for TA102 in the presence of S9 mix ŽTable 6.. 3.3. Induction of micronucleus response Micronucleus assay results are presented in Tables 7 and 8, which show the mean and standard deviation of frequency of PCE and micronucleated polychromatic erythrocytes ŽMNPCE., individually and by sex and groups. At the doses used, no toxicity in bone marrow was detected. The test groups were statistically similar to their respective negative control, when compared by the x 2-test. Based on these results, harman and harmine are probably not able to induce chromosome mutations in vivo.
4. Discussion Table 9 summarizes the results on the genotoxicity of the b-carboline alkaloids. The positive results to mutagenicity ŽTA97-S9. for harman and harmol
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
144
Table 6 Induction of Hisq revertants in S. typhimurium TA102 by b-carboline alkaloids, without ŽyS9. and with ŽqS9. metabolic activation Dose mgrplate
Substance
TA102 yS9 MI
NC d Harman
Response NC Harmine
10 20 30 40 50 60 80 100
0.9 1.1 1.1 1.2 1.3
e
5 10 15 20 25 40 60 80 100
1.1 1.2 1.4 1.6 1.6
10 20 30 40 50 60 80 100
1.0 1.0 1.0 1.0 0.9
Response
%S 100 98 97 70 67 69
c
MI
Revrplate
%S
305 " 3
100
1.1
320 " 4
99
1.1
342 " 6
99
1.2 1.3 1.2
371 " 4 384 " 28 376 " 8 "
92 90 76
238 " 16 250 " 29 297 " 11 323 " 5 375 " 14 391 " 9
100 89 81 71 72 41
380 " 29
100
1.2
469 " 27
103
1.3 1.5 1.3 1.5
486 " 34 552 " 5 496 " 45 588 " 23 "
81 73 65 49
323 " 25 337 " 35 337 " 17 330 " 13 311 " 4 297 " 10
100 95 83 77 80 59
275 " 21
100
1.0
285 " 18
84
1.1
293 " 23
85
1.1 1.1 1.0
315 " 17 298 " 28 279 " 3 y
86 81 85
290 " 18
100
1.1
306 " 28
84
1.0
295 " 21
59
1.0 1.1 1.3
300 " 19 321 " 23 380 " 23 "
61 59 38
360 " 18 387 " 18 392 " 23 395 " 33 407 " 26 412 " 18 y
100 72 80 96 86 63
y
50 100 150 200 250 300 400 500
1.0 1.0 1.0 0.9 0.8
Response NC Harmalol
280 " 15 251 " 35 304 " 11 321 " 17 325 " 15 356 " 16
b
"
Response NC Harmaline
TA102 qS9 Revrplate
"
Response NC Harmol
a
313 " 23 325 " 23 325 " 11 309 " 27 268 " 17 260 " 13
100 122 78 87 61 61
y
50 100 150 200 250
1.0 1.0 1.0 1.0 0.8
244 " 3 239 " 9 243 " 15 246 " 6 241 " 32 205 " 13 y
100 96 92 75 67 71
1.1 1.1 1.1 1.1 1.1
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
145
Table 7 Results of the micronucleus assay after a single treatment of male ŽM. and female ŽF. Swiss Webster mice with harman Group
a
Sex
Frequencies of polychromatic erythrocytes ŽPCE. in 1000 erythrocytes Mean " SD
Individual results Cq Cy T1 T2
M F M F M F M F
528 476 498 467 516 544 567 534
453 500 533 446 549 550 570 565
436 513 487 511 561 508 568 440
470 443 534 488 540 544 528 477
510 601 459 493 508 538 486 449
by sex
by group
479" 39 507" 59 502" 32 481" 25 535" 22 537" 17 544" 37 493" 54
493 " 49 492 " 29 536 " 19 518 " 51
Frequencies of micronucleated polychromatic erythrocytes ŽMNPCE. in 2000 PCE Mean " SD
Individual results Cq Cy T1 T2
M F M F M F M F
66 51 7 7 9 9 8 8
47 51 7 7 8 8 8 7
44 52 6 6 7 7 9 7
65 50 7 7 8 7 7 7
57 45 6 7 10 7 7 7
by sex
by group
55.8 " 1.0 49.8 " 2.8 6.6 " 0.5 6.8 " 0.4 8.4 " 1.1 7.6 " 0.9 7.8 " 0.8 7.2 " 0.4
52.8 " 7.7 6.7 " 0.5 8.0 " 1.1 7.5 " 0.7
a
Cq, positive control group; Cy, negative control group; T1 , test group killed 24 h after treatment; T2 , test group killed 48 h after treatment.
can be considered very weak frameshift mutation responses, with just over a two-fold increase in revertants over background observed in each case ŽTable 4.. In the SOS chromotest, only one positive result was found; harman weakly induced SOS functions without S9 mix, while the other related alkaloids showed non-induction or false-induction SOS Žsee Tables 1 and 2.. Eight signs of positivity results ŽTable 9. were found in TA102 as well as in frameshift strains TA98 and TA97, indicating marginal mutagenic activities in those cases. The negative results in TA100 indicated that these chemicals cannot be mutagens by base-pair substitution. Furthermore, the chemicals tested in the micronu-
cleus assay, harman and harmine, did not induce micronuclei in bone marrow PCE, suggesting that they cannot cause in vivo mutagenicity. We observed that the cytotoxicity of the alkaloids was reduced in the testing strains due to the use of S9 mix in the Salmonellarmicrosome assay and in the SOS chromotest as well. Harman, for instance, may be tested even at 250 mgrplate in the presence of S9 mix ŽTable 3. while in the absence of metabolization the largest dose was 50 mgrplate. Harmine at the concentrations of 10 and 100 mgrtest without S9 mix was cytotoxic in the SOS chromotest, which can be verified by the slope of UPase ŽTable 1., but this effect was not observed in the presence of
Notes to Table 6: a Mutagenic index: no. of Hisq induced in the samplerno. of spontaneous Hisq in the negative control. b Number of Hisqrplate: mean values at least of two experiments" SD. c Percent survival calculated in relation to the negative control: - 70% toxic dose. d Negative control: sterile distilled water Žharmine.; DMSOrsterile distilled water Ž1:1. Žharman, harmol, harmaline, and harmalol.. Positive control: ŽyS9. 4-nitroquinoline 1-oxide Ž0.5 mgrplate., 2477 " 180; ŽqS9. aflatoxin B1 Ž1 mgrplate., 1013 " 272. e y, Negative; q, positive ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI G 2.; ", signs of positivity ŽANOVA, p F 0.05; dose–response curve, p F 0.01; MI - 2..
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
146
Table 8 Results of the micronucleus assay after single treatment of male ŽM. and female ŽF. Swiss Webster mice with harmine Group
a
Sex
Frequencies of polychromatic erythrocytes ŽPCE. in 1000 erythrocytes Mean " SD
Individual results Cq
M F M F M F M F
Cy T1 T2
528 476 463 512 545 503 272 515
453 500 434 492 458 451 436 535
436 513 471 519 560 585 426 323
470 443 450 600 440 421 418 395
510 601 357 465 548 585 427 580
by sex
by group
479" 39 507" 59 435" 46 518" 51 510" 57 509" 75 396" 70 470" 107
493 " 49 476 " 63 510 " 63 433 " 94
Frequencies of micronucleated polychromatic erythrocytes ŽMNPCE. in 2000 PCE Mean " SD
Individual results Cq
M F M F M F M F
Cy T1 T2
66 51 6 6 11 7 6 7
47 51 7 6 7 7 7 7
44 52 7 5 7 9 7 6
65 50 6 6 5 7 7 7
57 45 6 7 8 7 7 9
by sex
by group
55.8 " 1.0 49.8 " 2.8 6.4 " 0.5 6.0 " 0.7 7.6 " 2.2 7.4 " 0.9 6.8 " 0.4 7.2 " 1.1
52.8 " 7.7 6.2 " 0.6 7.5 " 1.6 7.0 " 0.8
a
Cq, positive control group; Cy, negative control group; T1 , test group killed 24 h after treatment; T2 , test group killed 48 h after treatment.
metabolization and at the same concentrations ŽTable 2.. The same phenomenon occurred in the test with harmaline at 100 mgrtest ŽTables 1 and 2.. These effects of S9 mix can be explained by a partial detoxification of the alkaloids by reaction with microsomal enzymes of the S9 mix, such as epoxide
hydrolase and cytochrome P-450 mixed function oxygenase system, so that higher doses can be applied w33x. The recent review by Quillardet and Hofnung w23x determined that the correspondence between the SOS chromotest and Salmonellarmicrosome assay results
Table 9 Summary of results on the genotoxicity of the b-carboline alkaloids Alkaloid
Results
a
Mutagenicity TA98
Harman Harmine Harmol Harmaline Harmalol a
SOS induction TA97
TA100
TA102
MN induction
PQ37
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
y y y y y
y " y y y
q y q y y
" " y y y
y y y y y
y y y y y
" " y y y
" " y " y
q y y y y
y y y y y
y y NT NT NT
MN, micronuclei; yS9, without metabolic activation; qS9, with metabolic activation; q, positive; y, negative; ", signs of positivity; NT, not tested.
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
is very high Žover 82%.. Indeed, the induction of b-galactosidase synthesis in the SOS chromotest reflects the level of expression of SOS functions involved in cell division inhibition Ž sulA
147
ing harmol w36,38,39x. In vivo, harmol takes part in reactions of conjugation with glycuronic acid andror sulfate, but in vitro these reactions are poorly operative resulting in the free form w36,40x. Since harmol demonstrated mutagenic activity for TA97 in the direct test ŽTable 4., we may conclude that the mutagenicity obtained in the harmine tests on TA97 with S9 mix may be related to the formation of free harmol in situ. On the other hand, in the micronucleus assay, the putatively formed harmol in harmine-treated mice may have taken part in conjugating reactions resulting in reduced toxicity and lowered effects on the induction of micronuclei. In recent studies w11x, harman, harmine, harmaline, and norharman have been shown to be non-clastogenic in vitro for Chinese hamster ovary cells ŽCHO. in the presence and in the absence of S9 mix. Our results obtained from the micronucleus assay ŽTables 7 and 8. thus confirm in vivo that harman and harmine are not clastogenic as the MN frequency is not significantly increased in mouse PCE. Harman and harmine showed signs of positivity in TA102 with and without metabolic activation, whereas harmaline had the same type of response only in the presence of S9 mix ŽTable 6.. 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 alkylating agents, and has an intact excision–repair pathway w32x. Thus, these signs of positivity in TA102 can be due to DNA adduct formations by the alkaloids or by the metabolites generated by the S9 mix action in the A–T sites of bacterial DNA. Indeed, when harman was administered in the diet of mice, DNA adducts were found in liver and kidney w15x. As reported elsewhere w16,17x, aromatic b-carbolines, such as harman and harmine ŽFig. 1A., interact with DNA by intercalation. The same does not hold for the dihydro-b-carbolines, such as harmaline ŽFig. 1B.. Apparently this is due to the non-planarity of the ring system and the lack of full aromaticity of these dihydro-b-carbolines w17,41,42x. Therefore the activities reported here could be associated with the different abilities of these chemicals to interact with DNA. In fact, we showed that in the bacterial tests, the aromatic b-carbolines were more cytotoxic than the dihydro-b-carbolines. In addition to that, harman, harmine, and harmol, which have a totally aromatic
148
J.N. Picada et al.r Mutation Research 379 (1997) 135–149
structure ŽFig. 1A., showed a weak genotoxic activity or just signs of positivity, while harmaline and harmalol ŽFig. 1B. behave practically as non-genotoxic ŽTable 9.. Interestingly enough, these responses can be related to the degree of aromaticity and planarity of these compounds, inasmuch as observed in the studies on their ability to interact with DNA. It is interesting to note that in the micronucleus assay ŽTables 7 and 8., harman and harmine, which had shown to be the most active alkaloids in the bacterial tests, showed results indicating that they do not induce in vivo mutagenicity. The present results may suggest that these alkaloid b-carbolines probably do not carry long-term genetic risk, although further support is necessary to decide whether those chemicals can be hazardous to human health. Their biological effect still cannot be fully understood, such as that related to their comutagenic activity w8–12x. Furthermore, humans are sufficiently exposed to some of those chemicals, which are both present in plants used for the preparation of hallucinogenic material and medicinal drugs, and in tobacco smoke and well-cooked food w1–5x. As a consequence of these observations, further studies on the genetic toxicology of b-carbolines in other test systems would be worthwhile.
Acknowledgements We thank Drs. Martin Brendel, Tatiana Golubkova and Nikita Khromov-Borisov for helpful reading and discussion of the paper. This work was supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Cientıfico e ´ ŽCNPq., Fundac¸ao Tecnologico ´ ˜ de Amparo a` Pesquisa do Estado do Rio Grande do Sul ŽFAPERGS., and Laboratorio de Genotoxicidade ´ ŽGENOTOX.rCentro de BiotecnologiarUFRGS.
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