Mutagenesis vol. 22 no. 4 pp. 293–302, 2007 doi:10.

1093/mutage/gem016
Advance Access Publication 1 June 2007

Antioxidant properties of b-carboline alkaloids are related to their antimutagenic

and antigenotoxic activities

Dinara Jaqueline Moura1, Marc Francxois Richter2, Jane including monoamine oxidase inhibition (10–12), binding to
Marlei Boeira3, João Antonio Pêgas Henriques1,4 and benzodiazepine, serotonin, dopamine and imidazoline receptors
Jenifer Saffi1,4,* (13–17), convulsive or anticonvulsive actions, anxiolytic,
1
Departamento de Biofı́sica/Centro de Biotecnologia, Universidade Federal do tremorogenic and immunomodulatory effects (18–20). They
Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil, 2Laboratório de are also DNA intercalating agents (21,22) and inhibit enzymes,
Farmacocinética, Centro de Pesquisa em Ciências Médicas, Universidade e.g. DNA topoisomerases (23). Moreover, toxic and mutagenic
Luterana do Brasil (ULBRA), Canoas, RS, Brasil, 3Departamento de Ciências effects of these alkaloids have been reported in prokaryotic and
da Saúde, Universidade Regional Integrada do Alto Uruguai e das Missões
(URI), Erechim, RS, Brasil and 4Laboratório de Genética Toxicológica,
eukaryotic cells. These b-carboline alkaloids induce mutagenic
Universidade Luterana do Brasil (ULBRA), Canoas, RS, Brasil effects in various organisms such as Salmonella typhimurium
(24–26), Escherichia coli (25,26), Saccharomyces cerevisiae (8),

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The b-carboline alkaloids found in medical plants and in a V79 Chinese hamster lung cells (27,28) and human peripheral
variety of foods, beverages and cigarette smoke have a lymphocytes (29). However, alkaloids decreased the frequency
range of action in various biological systems. In vitro of cell damage when associated with several mutagenic agents
studies have demonstrated that these alkaloids can act as (30,31). They were also unable to induce significant genotoxic
scavengers of reactive oxygen species. In this paper, we effects in the same organism where positive results were observed
report the in vivo antioxidative properties of the aromatic (8,26,28,32).
(harmane, harmine, harmol) and dihydro-b-carbolines Some reports indicate that b-carbolines have effective anti-
(harmaline and harmalol) studied by using Saccharomyces oxidant properties. In this respect, harmane, harmaline and
cerevisiae strains proficient and deficient in antioxidant harmalol showed antioxidant activity by inhibiting lipid per-
defenses. Their antimutagenic activity was also assayed in oxidation in microsomal hepatic preparation (9) and by attenu-
S. cerevisiae and the antigenotoxicity was tested by the ating oxidative damage of hyaluronic acid, cartilage collagen
comet assay in V79 cell line, when both eukaryotic systems and immunoglobulin G (33,34). In addition, the biological
were exposed to H2O2. We show that the alkaloids have significance of b-carbolines has been related to their neuro-
a significant protective effect against H2O2 and paraquat
protective actions. Maher and Davis (35) demonstrated that
oxidative agents in yeast cells, and that their ability to
b-carboline alkaloids protect neurons against the excytotoxic
scavenge hydroxyl radicals contributes to their antimuta-
effect on dopamine and glutamate. Besides, other studies show
genic and antigenotoxic effects.
that these alkaloids exert a protective effect on oxidative neu-
ronal damage through a scavenging action on reactive oxygen
species (ROS) (36–38).
It is very well known that free radicals or ROS are respon-
Introduction sible for oxidative stress that can initiate physiopathological
The b-carbolinic alkaloids are widely distributed, being found processes, as age-related and chronic diseases like diabetes,
in several plant families, such as Apocynaceae, Elaeagnaceae, neurodegenerative and cardiovascular diseases, inflammation,
Leguminosae, Passifloraceae and Zygophyllaceae (1). They are Alzheimer and Parkinson’s disease and mainly carcinogenesis,
also found in cigarette smoke, overcooked foods and wine which occurs in a cell or in a tissue when ROS concentration
(2–5). Certain b-carbolines, such as harman, have been reported exceeds the antioxidant capability of that cell (39–41). As
as normal constituents of human tissues and body fluids (6). a consequence, much research has focused on antioxidants and
Other b-carbolines, like harmine and harmaline, are responsible on their action mechanisms. In line with this, several plant
for reported hallucinogenic effects of ‘ayashuasca’, a beverage extracts or secondary metabolites have been found to show
prepared with Banisteriopsis caapi and Pyschotria viridis, used strong antioxidant activity and protection against oxidant-
for religious purposes in South America and Africa (7,8). induced damage (42,43).
The metabolic pathway leading to the formation of b-carbolines In view of the fact that ROS are largely involved in DNA
is via Pictet–Spengler condensation between an indolamine (e.g. damage and mutagenesis, and that b-carboline alkaloids show
tryptamine) and aldehydes (e.g. acetaldehyde) (9). The common an antioxidant potential, it was interesting to evaluate its anti-
chemical structure of the alkaloids used in this study comprises mutagenic and antigenotoxic effects in different biological
one indole nucleus and one six-member pyrrole. According to models and to determine the concentration threshold of these
their oxidation state, these alkaloids can be divided into two effects. Therefore, the aim of the present study was to eval-
groups: dihydro-b-carbolines (harmaline and harmalol) and b- uate the antioxidant and antimutagenic/antigenotoxic properties
carbolines (harmane, harmine and harmol) (Figure 1). of harmine, harmane, harmol (fully aromatic), harmaline and
The b-carbolines have a wide spectrum of action, especially on harmalol (dihydro-b-carboline) alkaloids, and to correlate these
muscular, cardiovascular and central nervous systems (CNSs), biological responses to their chemical structure. We have used
*To whom correspondence should be addressed. Laboratório de Genética Toxicológica, Avenida Farroupilha 8001, Prédio 01 sala 122; Bairro São José,
CEP 92425-900, Canoas, RS, Brasil. Tel: þ55 51 34774000; Fax: þ55 51 34779214; Email: jenifer.saffi@ulbra.br

Ó The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org. 293
D. J. Moura et al.

H2O2 and paraquat to induce oxidative damage in S. cerevisiae oxide (DMSO) and distilled water; the final concentration of DMSO in the
strains defective in several antioxidant defenses. We have incubation mixture never exceeds 0.2%. The appropriate concentrations were
obtained by dilution of stock solutions in distilled water. The solvent controls
also evaluated the mutagenicity and antimutagenicity of the included in the genetic tests were found to be negative; 4-NQO, MMS, paraquat
alkaloids using the yeast strain N123, as well as their protective and H2O2 were used as positive control.
effect against oxidative DNA damage, verified by the comet
Medium and strains of S. cerevisiae
assay in a culture of permanent lung fibroblast cell line derived
The relevant genotypes of S. cerevisiae strains used in this study are listed in
from Chinese hamsters. Table I. Media, solutions and buffers were prepared as previously described
(44). The complete medium (YPD) containing 0.5% yeast extract, 2% peptone
and 2% glucose was used for routine growth. For plates, the medium was
Materials and methods solidified with 2% bacto-agar. The minimal medium (MM) contained 0.67%
Chemicals yeast nitrogen base without amino acids, 2% glucose and 2% bacto-agar. The
synthetic complete medium (SC) was MM supplemented with 2 mg adenine,
The alkaloids harmane (CAS 21655-84-5), harmine (CAS 343-27-1), harmol 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg uracil and
(CAS 149022-16-2), harmaline (CAS 6027-98-1) and harmalol (CAS 6028-07-5) 2 mg tryptophan per 100 ml MM. For mutagenesis, plates were supplemented
hydrochlorides, H2O2, paraquat (methyl viologen), methyl methanesulfonate with 60 lg/ml canavanine (SC þ can).
(MMS), 4-nitroquinoline-N-oxide (4-NQO), hypoxanthine, xanthine oxidase and We chose to work in the stationary phase of growth because this resembles
salicylic acid were obtained from Sigma (St Louis, MO, USA). Dulbecco’s most cells of multicellular organisms in important aspects: (i) most energy
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin–ethy- comes from mitochondrial respiration, (ii) the cells have left the active cell
lenediamine tetraacetic acid (EDTA), L -glutamine, antibiotics and trypan blue cycle and have entered the G0 phase and (iii) damage accumulates over time
were purchased from Gibco BRL (Grand Island, NY, USA). Low-melting (45,46). The herbicide paraquat, a redox cycling compound, was used to in-
point agarose (LMA) and agarose were obtained from Invitrogen (Carlsbad, CA, crease the intracellular flux of superoxide anion (O2–.). The appropriate concen-

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USA). Yeast extract, bacto-peptone and bacto-agar were obtained from Difco trations of H2O2 and paraquat were determined by survival assay, according
Laboratories (Detroit, MI, USA). All others reagents were of analytical grade. to the differential sensitivity of each strain. Sub-lethal concentration of the
For treatment of cells, 5 mg/ml stock solutions of the alkaloids were pre- oxidants was used for all subsequent experiments.
pared immediately prior to use. Harmine was dissolved in distilled water and
harman, harmol, harmaline and harmalol were dissolved in 5% dimethylsulf- Survival assays in S. cerevisiae strains
Stationary phase cultures of EG103 [wild type (WT)] and mutant isogenic strains,
as well as YPH98 (WT) and the isogenic mutant strain, were obtained by in-
oculation of an isolated colony into liquid YPD. To evaluate sensitivity to b-
carboline alkaloids, cultures were exposed to concentrations varying from 25 to
150 lg/ml and incubated under growth conditions for 1 h in phosphate-buffered
saline (PBS) at 30°C. Cells were appropriately diluted and plated in triplicate
on solid YPD (2–3 days, 30°C) after colony-forming units were counted.
To verify the antioxidant activity of the alkaloids, cells were pre-treated in
PBS with non-cytotoxic concentrations of alkaloids and incubated for 1 h at
30°C. Cells were then washed and treated with paraquat or H2O2 in PBS for
another hour. For survival determination, suitable aliquots were plated in
triplicate on solid YPD. Plates were incubated at 30°C for 2–3 days before
counting the colonies. All tests were repeated at least 3-fold, and plating was
carried out in triplicate for each dose.

Detection of forward mutation and potential antimutagenic activity in

S. cerevisiae
Saccharomyces cerevisiae N123 strain was used for assaying alkaloid mutage-
nicity as well as the protective effect of the alkaloids against H2O2-induced
mutagenesis. This strain was chosen because it is very responsive to H2O2-
induced mutagenesis due to its low glutathione content (47). A suspension of
2
108 cells/ml in the stationary phase, grown in YPD (2% glucose), was
incubated for 1 h at 30°C with various concentrations of alkaloids in PBS.
Survival was determined on SC (2–5 days, 30°C), and mutation induction
(CAN revertants) on appropriate supplementation media (4–5 days, 30°C).
Forward mutation was measured with the canavanine resistance assay (CAN1-
can1) after induction with different treatments. This assay uses a phenotypic
marker, canavanine sensitivity, since WT yeast strains express the arginine
transporter Can1p, which also imports canavanine from the environment and
leads to cell death (48). Thus, mutagen-induced alterations in the CAN1 gene
Fig. 1. Chemical structure of the b-carboline alkaloids. (A) Aromatic that impair Can1p functionality can increase cell survival in the presence of
b-carboline. (B) dihydro-b-carbolines. canavanine, when compared to a non-mutagenic cell sample.

Table I. Saccharomyces cerevisiae strains used in this study

Strain Genotype Enzymatic defense lacking Source

EG103 (SOD-WT) MATa leu2D0 his3-D1 trp1-289 ura3-52 None E. Gralla

EG118 (sod1D) Like EG103, except sod1::URA3 Cu–Zn SOD (cytosolic) E. Gralla
EG110 (sod2D) Like EG103, except sod2::TRP1 MnSOD (mitochondrial) E. Gralla
EG133 (sod1D sod2D) Like EG103, except sod1::URA3 e sod2::TRP1 All SOD E. Gralla
EG223 (ctt1D) Like EG103, except ctt1::TRP1 Cytosolic catalase E. Gralla
EG213 (sod1D ctt1D) Like EG103, except sod1::URA3 e ctt1::TRP1 Cu–Zn SOD and cytosolic catalase E. Gralla
YPH98 (WT) MATa ade2-101 leu2-D1 lys2-801 trp1-D1 ura3-52 None P. Hieter
yap1D Like YPH98 except yap1::URA3 yAP-1 transcription factor M. Grey
N123 MATa his1-7 None, but exhibits low glutathione content J. Henriques

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For antimutagenic evaluation, the procedure was as follows: cells were ybenzoic acid and 2,3-dihydroxybenzoic acid (DHBAs), produced by the
submitted to pre-treatment with non-cytotoxic concentrations of alkaloids and reaction of salicylic acid with produced hydroxyl radicals (OH_) was deter-
incubated for 1 h with shaking at 30°C. Cells were then washed and H2O2 was mined from standard curves of the respective dihydroxyphenols.
added. The mixture was further incubated at 30°C for 1 h. After treatment,
appropriate dilutions of cells were plated onto SC plates to determine cell Statistics
survival, and 100 ll aliquots of cell suspension (2
108 cells/ml) were plated Statistical analyses of the data were performed using one-way analysis of
onto SC media supplemented with 60 lg/ml canavanine. Plates were incubated variance (ANOVA)–Tukey’s multiple comparison test. P-values under 0.05
in the dark at 30°C for 3–5 days before counting the survivors and revertant were considered significant. Data were expressed as means  SDs values.
colonies. All mutagenicity assays were repeated at least three times, and plating
for each dose was conducted in triplicate.
Results
Comet assay using V79 cells
Chinese hamster lung fibroblasts (V79 cells) were cultivated under standard Protective effects of b-carbonilic alkaloids in S. cerevisiae
condition in DMEM supplemented with 10% FBS, 2 mM L -glutamine and strains
antibiotics (49). Cells were maintained in tissue culture flasks at 37°C in WT cells and isogenic mutant strains of S. cerevisiae lacking
a humidified atmosphere containing 5% CO2, and were harvested by treatment
with 0.15% trypsin and 0.08% EDTA in PBS. Cells (2
105) were seeded into antioxidant defenses (Table I) were treated with several con-
each flask and cultured 1 day prior to treatment. Alkaloids were added to FBS- centrations of the alkaloids for 1 h during the stationary phase.
free medium to achieve the different designed concentrations, and the cells All strains showed practically the same sensitivity for b-
were treated for 2 h at 37°C in a humidified atmosphere containing 5% CO2. carbolines to that observed for the WT cells (Figure 2). Our
Oxidative challenge with 0.1 mM H2O2 was carried out for 0.5 h in the dark in
FBS-free medium. The culture flasks were protected from direct light during
findings showed that harmane, harmine and harmol decrease
treatment with the alkaloids and H2O2. viability but, in a significant way, only in concentrations up to

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The alkaline comet assay was performed as described by Singh et al. (50) 150 lg/ml, whereas harmaline and harmalol do not induce
with minor modifications (51). At the end of treatment, cells were washed with significant effects in any of the concentrations employed.
ice-cold PBS and trypsinized with 100 ll trypsin (0.15%). Immediately In this manner, we chose non-cytotoxic alkaloid concentra-
thereafter, 20 ll of cell suspension (106 cells/ml) were dissolved in 0.75%
LMA and spread on normal agarose point (1%) pre-coated microscope slides. tions (ranging from 25 to 100 lg/ml) to follow experiments in
Cells were ice-cold lysed (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH order to verify the protecting activity against oxidants in the
10.0, with freshly added 1% Triton X-100 and 10% DMSO) for at least 1 h at same strains.
4°C in order to remove cellular proteins and membranes, leaving the DNA as To verify an intracellular protective effect of the alkaloids,
‘nucleoids’. Thereafter, slides were placed in a horizontal electrophoresis box,
containing freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA,
i.e. a possible role of b-carboline alkaloids in cell oxidative
pH 13.0) for 20 min at 4°C in order to allow DNA unwinding and expression stress, yeast cells were pre-treated with non-cytotoxic concen-
of alkali-labile sites. An electric current of 300 mA and 25 V (0.90 V/cm) was tration of harmane, harmine, harmol, harmaline or harmalol,
applied for 20 min to electrophorese DNA. All the steps above were performed and then further exposed to sub-lethal concentrations of either
under yellow light or in the dark in order to prevent additional DNA damage. H2O2 or paraquat. A statistically significant survival was ob-
Slides were then neutralized (0.4 M Tris, pH 7.5), stained with silver nitrate as
described by Nadin et al. (52) and analyzed using an optic microscope. Images served as a consequence of antioxidant effect of the alkaloids.
of 100 randomly selected cells (50 cells from each of two replicate slides) were The aromatic b-carboline harmane significantly enhanced
analyzed per group. Cells were also scored visually into five classes, according the survival of all yeast cells, with EG103 background at 50
to tail size (from undamaged, 0, to maximally damaged, 4). and 100 lg/ml after treatment with H2O2 (Figure 3A), showing
International guidelines and recommendations for the comet assay consider
that visual scoring of comets is a well-validated evaluation method (53). It has
a clear antioxidant protective effect. However, after treatment
a high correlation with computer-based image analysis. The damage index (DI) with paraquat, this effect was less significant, being more
is based on the length of migration and on the amount of DNA in the tail and is effective at 50 lg/ml in these strains (Figure 4A). Although
considered a sensitive measure of DNA and of damage frequency (DF), as the harmane did not protect YPH98 WT against any of the two
proportion of cells that show tails after electrophoresis. Image length (IL) or oxidative agents, an important antioxidant effect was observed
migration length gives information only about the size of DNA fragments, and
is largely dependent upon electrophoresis conditions (i.e. voltage and duration). against H2O2 for yap1D, as verified by the increase in survival
Thus, DI and DF are emphasized in our analyses. The other parameter, IL, after oxidative challenge and shown in Figure 3A.
though considered in the analysis, was used only as a complementary DNA Figure 3B shows that harmine at 50 lg/ml was able to
damage parameter. DI was thus assigned to each comet according to its class, protect sod1D, sod2D and ctt1D single mutants against H2O2
and ranged from 0 (completely undamaged: 100 cells
0) to 400 (with max-
imum damage: 100 cells
4) (54). The DF (%) was calculated as the number
cytotoxicity. In addition, this activity was more effective in the
of cells with tails versus those without (0–100%). Results are presented sod1Dctt1D double mutant. However, this alkaloid did not
as means and ranges of four independent experiments. The solvent was used as protect any yeast strain against the deleterious effects of para-
negative control; MMS (4
105 M) and H2O2 (0.1 mM) were used as pos- quat (Figure 4B).
itive control. After treatment with H2O2, the harmol antioxidant activity
was only observed for the strains deficient in both superoxide
Hipoxanthine/xanthine oxidase assay
dismutases (SODs) (single and double mutants) and in the
The method employed to assay the hydroxyl radical-scavenging ability of
alkaloids was based on the method of Owen et al. (55). Briefly, alkaloids were
transcription factor-deficient mutant yap1 (Figure 3C). How-
dissolved in the assay buffer [hypoxanthine, Fe(III), EDTA and salicylic acid] ever, harmol protected EG103 WT as well as sod1D, ctt1D
at a concentration of 2.0 mg/ml and diluted appropriately (in triplicate) in assay single and sod1Dsod2D, sod1Dctt1D double mutants against
buffer to a final volume of 1.0 ml giving a range of 0.5–1.5 mg/ml. A 5-ll treatment with paraquat (Figure 4C).
aliquot of xanthine oxidase dissolved in 3.2 M (NH4)2SO4 was added to initiate Harmaline (Figures 3D and 4D) showed a significant pro-
the reaction. The sample tubes were incubated at 37°C for 3 h, at which time
the reaction was complete. A 30-ll aliquot of the reaction mixture was analyzed tection against H2O2 and paraquat, respectively, although this
by high-pressure liquid chromatography (HPLC) using chromatographic con- effect is more prominent in the single sod-deficient mutants.
ditions as previously described (56). Chromatographic analysis was done using Dihydro-b-carboline harmalol demonstrated the strongest
a gradient based on methanol–water–acetic acid with a lBondaPak C18 reverse antioxidant effect (Figures 3E and 4E). This alkaloid signif-
phase column (Waters) and detection at 325 nm. The HPLC equipment had a
2695 separation module (Waters) and UV detector 2487 (Waters). The hydrox-
icantly enhanced the survival of yeast cells at 50 and 100 lg/ml
ylation of salicylic acid and hypoxanthine was monitored at A 5 325 and for EG103 WT and its sod isogenic mutant strains (sod1D,
A 5 278 nm, respectively. The amount of dihydroxyphenol, 2,5-dihydrox- sod2D and sod1Dsod2D) in the pre-treatment assay, using

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Fig. 2. Sensitivity of cells in the stationary growth phase to harmane (A), harmine (B), harmol (C), harmaline (D) and harmalol (E). EG103 (WT) (filled square) and
its isogenic derivative strains: sod1D (filled diamond), sod2D (filled circle), sod1Dsod2D (filled triangle), ctt1D (open diamond), sod1Dctt1D (open circle), YPH98
(WT) (open square) and isogenic strain yap1 (open triangle). Cells were treated for 1 h at 30°C.

H2O2 (Figure 3E) and paraquat (Figure 4E) as oxidants. Similar in the presence (Table IV) and absence (Table V) of H2O2. We
results were obtained for the mutant lacking the transcription evaluated the genotoxic effect in this cell line as well as its
factor Yap1p (yap1D). antigenotoxic properties. Table IV shows that aromatic b-
carbolines alkaloids (harmane, harmine and harmol) induced
Induction of forward mutation and antimutagenic effects in S. DNA damage, as verified by DI and DF increase at the highest
cerevisiae concentration employed (40 lg/ml). On the other hand, dihydro-
The b-carboline alkaloids did not induce mutagenic effect in b-carbolines (harmaline and harmalol) did not generate signif-
stationary growth phase in S. cerevisiae N123 strain (Table II). icant DNA damage at the concentration range evaluated.
Once again, we chose a non-cytotoxic alkaloid concentration H2O2-induced DNA damage was used to check any possible
(10–50 lg/ml) to follow experiments in order to verify the pro- antigenotoxic effect of these alkaloids. As expected, exposure
tective effects of b-carbolinic alkaloids against the H2O2- of V79 cells to H2O2 resulted in a significant increase in DNA
induced forward mutagenesis in N123 yeast strain. Table III damage parameters DI and DF (Table V). b-Carboline pre-
shows that all b-carbolines inhibited the mutagenic action of treatment at lower concentrations significantly inhibited the
H2O2, mainly by an increase in cell survival. Dihydro-b- DNA damage induced by this agent, reducing the DI and DF.
carboline harmalol had the most prominent effect, increasing Harmane, harmine and harmol showed a significant decrease in
the survival during H2O2 treatment and simultaneously decreas- the DI and DF at lower concentrations (10 and 20 lg/ml), in
ing induced mutation in yeast. comparison to the DNA-damaging effects of H2O2 (Table V).
On the other hand, harmalol and harmaline clearly demon-
Comet assay strated a significant reduction in DI and DF in a large concen-
The effects of all alkaloids on DI and DF, as measured by DNA tration range (10–40 lg/ml). This decrease of damage score
damage in V79 cells, according to the comet assay, are shown does not occur in a dose-dependent manner and is similar for

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Fig. 3. Effect of pre-treatments with b-carbolines on survival after treatment with oxidant H2O2 in EG103 (WT) and mutant isogenic strains and YPH98 (WT) and
mutant isogenic strains. (A) H2O2 (5 mM) (black bars), 50 lg/ml harmane þ H2O2 (5 mM) (gray bars) and 100 lg/ml harmane þ H2O2 (5 mM) (white bars). (B)
H2O2 (5 mM) (black bars), 50 lg/ml harmine þ H2O2 (5 mM) (gray bars) and 100 lg/ml harmine þ H2O2 (5 mM) (white bars). (C) H2O2 (5 mM) (black bars), 50
lg/ml harmol þ H2O2 (5 mM) (gray bars) and 100 lg/ml harmol þ H2O2 (5 mM) (white bars). (D) H2O2 (5 mM) (black bars), 50 lg/ml harmaline þ H2O2 (5
mM) (gray bars) and 100 lg/ml harmaline þ H2O2 (5 mM) (white bars). (E) H2O2 (5 mM) (black bars), 50 lg/ml harmalol þ H2O2 (5 mM) (gray bars) and 100 lg/
ml harmalol þ H2O2 (5 mM) (white bars). Percentage survival is expressed relative to the untreated control culture (100%). Values shown are the mean at least three
determinations. Data significant in relation to oxidant-treated samples at *P , 0.05, **P , 0.01 and ***P , 0.001/one-way ANOVA–Tukey’s multiple
comparison test.

all concentrations used. This response is also very interesting of DHBA. In this manner, b-carbolines showed a significant
since it shows that a significant antigenotoxic effect can be antioxidant capacity in a dose-dependent manner at high con-
reached by using low concentrations of the alkaloids. The centrations due the compounds’ hydroxyl radical-scavenging
frequency of damage class was different for each alkaloid for ability.
each dose.
In vitro antioxidant capacity of b-carboline alkaloids Discussion
The antioxidant capacity of b-carboline alkaloids was deter- b-Carboline alkaloids are active constituents of hallucinogenic
mined by monitoring the production of hydroxyl benzoic acids plants used in South American Indian cultures (7) and have
(DHBA) due to the attack of ROS on salicylic acid in the been identified in plants that have a long tradition in ethno-
hypoxanthine–xanthine oxidase assay. The reduction of total pharmacology. Pharmacological investigations on the alkaloids
oxidation products as a function of the concentration of alka- have demonstrated interesting biological activities, including
loids added to the assay is shown in Figure 5. All b-carboline the inhibition of monoaminoxidase, binding to a wide range of
alkaloids demonstrated a significant antioxidant capacity in CNS receptors and anxiolytic and tremorogenic effects (11–
a dose-dependent manner. Harmane, harmalol and harmaline 20). Furthermore, in vitro studies show antioxidative and
had a more pronounced activity, reducing the formation of both neuroprotective actions of the b-carboline alkaloids (9,33–38).
DHBA species to 2.7, 7.15 and 8.72%, respectively, in the In this manner, our interest was placed on the evaluation of the
highest concentration used (1.5 mg/ml), whereas harmol (38.2%) antioxidant and antimutagenic/antigenotoxic effects of these
and harmine (42.2%) showed moderate activity in the reduction molecules on yeast defective in antioxidant defenses and in

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Fig. 4. Effect of pre-treatments with b-carbolines on survival after treatment with oxidant paraquat in EG103 and mutant isogenic strains and YPH98 (WT) and
mutant isogenic strains. (A) Paraquat (5 mM) (black bars), 50 lg/ml harmano þ paraquat (5 mM) (gray bars) and 100 lg/ml harmano þ paraquat (5 mM) (white
bars). (B) Paraquat (5 mM) (black bars), 50 lg/ml harmine þ paraquat (5 mM) (gray bars) and 100 lg/ml harmine þ paraquat (5 mM) (white bars). (C) Paraquat (5
mM) (black bars), 50 lg/ml harmol þ paraquat (5 mM) (gray bars) and 100 lg/ml harmol þ paraquat (5 mM) (white bars). (D) Paraquat (5 mM) (black bars), 50
lg/ml harmaline þ paraquat (5 mM) (gray bars) and 100 lg/ml harmaline þ paraquat (5 mM) (white bars). (E) Paraquat (5 mM) (black bars), 50 lg/ml
harmalol þ paraquat (5 mM) (gray bars) and 100 lg/ml harmalol þ paraquat (5 mM) (white bars). Percentage survival is expressed relative to untreated control
culture (100%). Values shown are the means of at least three determinations. Data significant in relation to oxidant-treated samples at *P , 0.05, **P , 0.01 and
***P , 0.001/one-way ANOVA–Tukey’s multiple comparison test.

V79 cell line, in the first study to investigate these alkaloids genetic background, and also influences the response to the
as potential protective agents using H2O2 and paraquat as alkaloids treatment. The results in yeast survival tests, employing
oxidative agents. the EG103 isogenic strains sod1D, sod2D, ctt1D, sod1Dctt1D
In this sense, we demonstrate that b-carboline alkaloids show and sod1Dsod2D, pre-treated with these alkaloids, showed a
a protective effect against oxidative agents using yeast and general survival increment after exposure to H2O2 (Figure 3) and
mammalian cells as two eukaryotic model organisms. The paraquat (Figure 4). Furthermore, the dihydro-b-carboline
antioxidative effect observed for harmane, harmine, harmol, alkaloids (harmalol and harmaline) had a higher protective effect
harmaline and harmalol clearly depends on the structure and as compared to the aromatic b-carbolines (harmane, harmine and
concentration of the alkaloid. harmol). The antioxidant effect was more pronounced for H2O2,
The yeast S. cerevisiae has been a useful model for studies which may suggest that the alkaloids are acting as scavengers of
of the eukaryotic response to oxidant challenge (46). In this hydroxyl radicals (OH_), generated through the Haber-Weiss–
study, we have used yeast strains null mutant in the cytosolic Fenton reaction (57). It is important to take into account that the
CuZnSOD gene (sod1D strains), mitochondrial MnSOD gene superoxide anion (generated by paraquat treatment) is known to
(sod2D strains), cytosolic catalase gene (ctt1D strain) and double oxidize exposed (4Fe-4S) clusters in certain enzymes, leading to
mutants (sod1Dsod2D and sod1Dctt1D). Besides, null mutant in inactivation of the enzyme and liberation of iron (58,59), which
yAP-1 transcription factor was also used. The H2O2 and paraquat thus becomes available to participate in the Fenton reaction, and
concentrations used in the assays were appropriate for the consequently yields OH_ radicals. In addition, Bayliak et al. (60)
differential sensitivity of each strain, which is dependent on their suggest that SOD play an important role in yeast survival under

298
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Table II. Induction of forward mutation (can1) in haploid N123 strain of Table IV. Effects of b-carbolines alkaloids in V79 cells exposed for 2 h and
Saccharomyces cerevisiae after b-carbolinic alkaloids treatments in stationary evaluated by comet assay
phase in PBS
Substance Treatment DIa DF (%)a
Agent Treatment (lg/ml) Survival (%) Can/107 survivorsa
NCb 0 46.00  4.00 49.00  2.00
NCb 0 100 (237)c 1.05  0.57d MMSc 4.0
105 M 227.00  4.35*** 86.00  9.84***
4NQOe 0.5 45.14 (107)*** 30.28  3.43*** Harmane 10 lg/ml 80.00  8.93 41.33  3.05
Harmane 10 98.78 (234) 2.54  0.02 20 lg/ml 84.33  3.79 58.00  3.00
25 91.71 (217) 1.98  1.03 40 lg/ml 182.71  6.19** 69.66  1.57*
50 87.23 (207) 2.55  0.42 Harmine 10 lg/ml 74.00  16.28 54.33  4.61
Harmine 10 96.55 (228) 1.71  0.45 20 lg/ml 81.01  0.73 65.66  6.42
25 93.13 (221) 1.39  0.52 40 lg/ml 113.2  4.58* 71.64  1.82*
50 90.72 (215) 2.77  0.36 Harmol 10 lg/ml 82.68  5.85 54.33  3.53
Harmol 10 90.71 (215) 1.90  0.59 20 lg/ml 80.02  13.89 56.00  2.00
25 88.30 (209) 2.08  0.42 40 lg/ml 103.33  7.57* 58.30  14.97
50 86.56 (205) 2.23  0.43 80 lg/ml 150.00  12.76*** 72.00  5.29**
Harmaline 10 96.63 (229) 1.55  0.29 Harmaline 10 lg/ml 49.66  20.42 35.66  10.96
25 95.47 (226) 1.65  0.98 20 lg/ml 66.34  11.93 50.60  7.8
50 93.96 (222) 2.03  0.30 40 lg/ml 80.67  10.42 52.62  4.72
Harmalol 10 92.26 (219) 1.25  0.75 80 lg/ml 79.00  13.51 54.00  7.20
25 91.49 (217) 1.97  0.28 Harmalol 10 lg/ml 55.38  6.65 48.66  10.96
50 89.23 (211) 2.03  0.24 20 lg/ml 58.66  4.60 50.00  4.35

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40 lg/ml 63.05  2.83 52.66  10.59
a
Locus-specific revertants. 80 lg/ml 62.33  15.53 53.65  8.62
b
Negative control (solvent).
c a
Number of colonies. Means values and standard deviation obtained from average of 100 cells per
d
Mean and standard deviation per three independent experiments. experiment—total of four experiments per dose for each substance.
e b
Positive control. Negative control (solvent).
c
Data significant in relation to negative control group (solvent) at Positive control.
***P , 0.001/one-way ANOVA–Tukey’s multiple comparison test. Data significant in relation to negative control (solvent) groups at *P , 0.05,
**P , 0.01 and ***P , 0.001/one-way ANOVA–Tukey’s multiple
comparison test.

Table III. Effects of b-carbolinic alkaloids on induced mutagenicity by H2O2

in haploid N123 strain of Saccharomyces cerevisiae in the stationary phase
in PBS
Table V. Effect of b-carboline alkaloids in V79 cells exposed for 2 h plus
Agent Treatment Survival (%) Can/107 survivorsa oxidant H2O2 for 0.5 h and evaluated by comet assay

NCb 0 100 (247)c 1.02  0.12d Substance Treatment DIa DF (%)a

H2O2e 4 mM 42.10 (104) 19.29  2.08
Harmane 10 lg/ml þ H2O2 74.89 (185) 5.67  0.80*** NCb 0 46.35  4.83 22.45  6.11
25 lg/ml þ H2O2 69.73 (172) 6.99  0.10** H2O2c 100 lM 219.66  36.08 86.36  6.35
50 lg/ml þ H2O2 67.20 (166) 9.64  1.81** Harmane 10 lg/ml þ H2O2 92.33  13.79*** 41.33  3.05***
Harmine 10 lg/ml þ H2O2 70.85 (175) 5.95  0.38*** 20 lg/ml þ H2O2 122.00  6.00*** 58.00  3.00**
25 lg/ml þ H2O2 60.42 (149) 7.49  1.11** Harmine 10 lg/ml þ H2O2 106.00  21.96*** 55.66  6.42**
50 lg/ml þ H2O2 55.46 (137) 13.5  1.57* 20 lg/ml þ H2O2 123.33  24.58* 67.33  2.30
Harmol 10 lg/ml þ H2O2 79.77 (197) 4.46  0.57*** Harmol 10 lg/ml þ H2O2 92.66  11.60*** 44.33  2.51***
25 lg/ml þ H2O2 72.82 (180) 4.93  0.41*** 20 lg/ml þ H2O2 108.66  19.03*** 56.33  2.00**
50 lg/ml þ H2O2 61.34 (152) 10.92  2.26* Harmaline 10 lg/ml þ H2O2 106.33  11.93*** 60.00  7.80***
Harmaline 10 lg/ml þ H2O2 65.47 (162) 4.44  0.22*** 20 lg/ml þ H2O2 107.66  7.57*** 50.33  15.71***
25 lg/ml þ H2O2 67.26 (166) 6.68  1.66** 40 lg/ml þ H2O2 106.00  1.73*** 54.33  4.61***
50 lg/ml þ H2O2 73.99 (183) 7.59  3.15** Harmalol 10 lg/ml þ H2O2 86.33  12.85*** 50.00  11.36***
Harmalol 10 lg/ml þ H2O2 68.70 (170) 8.88  1.30** 20 lg/ml þ H2O2 112.33  8.38*** 49.66  4.93***
25 lg/ml þ H2O2 74.89 (185) 4.38  0.32*** 40 lg/ml þ H2O2 115.33  17.00*** 54.66  3.51***
50 lg/ml þ H2O2 71.25 (176) 1.98  0.19***
a
Mean values and standard deviation obtained from average of 100 cells per
a
Locus-specific revertants. experiment—total of four experiments for each substance.
b b
Negative control (solvent). Negative control (solvent).
c c
Number of colonies. Positive control (H2O2).
d
Mean and standard deviation per three independent experiments. Data significant in relation to positive control (oxidant) group at *P , 0.05,
e
Positive control (H2O2). **P , 0.01 and ***P , 0.001/one-way ANOVA–Tukey’s multiple
Data significant in relation to positive control group at *P , 0.05, **P , 0.01 comparison test.
and ***P , 0.001/one-way–ANOVA Tukey’s multiple comparison test.
action, since there are evidences demonstrating that these
alkaloids are not active against superoxide anions in vitro when
oxidative stress induced by H2O2, and it has been shown that tested using SOD-inhibitable reduction ferricytochrome c (36).
there is a strong relationship between catalase and SOD activities It is also important to note the antioxidant response observed
under different experimental conditions. Therefore, the lack of in the yap1 mutants for most alkaloid treatments, especially for
SOD as well as of catalase activities imputes sensitivity to H2O2. the dihydro-b-carbolines (Figures 3D, 3E, 4D and 4E). Yap1
Thus, we believe that the protective effect against paraquat is a key regulator of oxidative stress tolerance in S. cerevisiae,
toxicity can be due to the direct action against H2O2 or against and has been shown to regulate a broad set of genes in response
OH_ radical generated through the Haber-Weiss–Fenton re- to oxidative stress, including TRX2 (thioredoxin), TRR1

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D. J. Moura et al.

the alkaloids against ROS, especially hydroxyl radicals, in the

following decreasing order: harmalol . harmaline . harmol
. harmane . harmine.
Indole precursors of the b-carbolines, tryptophan and trypt-
amines, are known to have antioxidative activities, possibly by
scavenging reactive oxygen radicals and forming a stable
indole radical at the pyrrole ring (69–71). Tse et al. (9) suggest
that the indole nucleus of the b-carbolines can have similar
antioxidant properties. This could explain the strong in vitro
antioxidant activity observed by harmane (in the xanthine
oxidase assay), which does not possess a ring substitution. How-
ever, since this alkaloid can intercalate in DNA (26,28,72,73),
this could also be responsible for the reduced antioxidant ac-
tivity observed in yeast cells, as well as for the reduced anti-
mutagenic and antigenotoxic activities in our experiments.
Our results reinforce what was described by Tse et al. (9): the
b-carboline antioxidative actions are dependent on chemical
Fig. 5. Inhibition of the generation of reactive oxygen species by b-carboline structure. Dehydrogenation of the pyridyl ring (e.g. harmalol
alkaloids in hypoxanthine–xanthine oxidase systems. Solvent (hexane) (filled to harmol, harmaline to harmine) resulted in a considerable

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square), harmane (filled triangle), harmine (filled circle), harmol (open square), decrease in antioxidant efficacies. The replacement of the
harmalol (open circle) and harmaline (open triangle).
hydroxyl group by a methoxyl group also decreases the anti-
oxidant effect (e.g. harmalol to harmaline, harmol to harmine).
(thioredoxin reductase), GLR1 (glutathione reductase) and Of all b-carbolines studied, harmalol was found to have the
GSH1 (c-glutamylcysteinesynthase) (61,62). Consequently, highest scavenger action.
yap1D mutants are sensitive to oxidative stress (62). Hence, In summary, our findings indicate that the b-carboline alkaloids
only potent antioxidants are able to protect this strain in this have a significant antioxidative effect in yeast and that their
model. Our results thus demonstrate a putative direct action of the hydroxyl radical-scavenging property appears to contribute to
alkaloids as ROS scavengers, rather than an induction of other their antimutagenic and antigenotoxic effects, observed in yeast
antioxidant defenses that would lead to an adaptive response and mammalian cells, respectively. Since no other data regard-
in yeast. ing the in vivo effects of harmane, harmine, harmol, harmaline
The capacity of b-carbolines to scavenge hydroxyl radicals and harmalol are available, further studies should be conducted
was confirmed in the results of the in vitro hypoxanthine– to define the antioxidant properties of these b-carbolines.
xanthine oxidase assay (Figure 5). In agreement with our
findings, Lee et al. (36) have shown that b-carbolines (harmalol,
harmaline and harmine) caused the decomposition of hydroxyl Acknowledgements
radicals, assayed by inhibition of 2-deoxy-D -ribose degradation, We thank Dr Cristine Gaylard, Dr Eloy J. Garcia and Ms Renato Moreira Rosa
and that the dihydro-b-carbolines harmalol and harmaline pro- for the critical reading of the manuscript. Research was supported by grants from
duce the most effective activity. the Brazilian Agencies ‘Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico’ (CNPq), ‘Fundac xão de Amparo a Pesquisa do Rio Grande do Sul’
ROS-induced DNA damage may cause mutations resulting (FAPERGS) and GENOTOX-Laboratory of Genotoxicity—Royal Institute,
in neurodegenerative disease, cancer and ageing (41,63–65). Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Oxidative lesions in DNA include base modifications, sugar
damage, strand breaks and abasic sites. H2O2 causes strand
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Received on February 6, 2007; revised on March 23, 2007;

accepted on April 2, 2007

302