Orbital: The Electronic Journal of Chemistry
journal homepage: www.orbital.ufms.br
ISSN 1984-6428
| Vol 9 | | No. 1 | | January-March 2017 |
Full Paper
Antigenotoxicity of Depsidones Isolated from Brazilian Lichens
Zaira da Rosa Guterresa, Neli Kika Hondab, Roberta Gomes Coelhob, Glaucia Braz
Alcantarab, Ana Camila Michelettib*
a
Universidade Estadual de Mato Grosso do Sul, Unidade Universitária de Mundo Novo, BR 163, km 202;
Mundo Novo, MS 79980-000, Brazil.
b
Instituto de Química, Universidade Federal de Mato Grosso do Sul, Av. Senador Filinto Müller, 1555; Campo
Grande, MS 79074-460, Brazil.
Article history: Received: 24 August 2016; revised: 09 March 2017; accepted: 17 March 2017. Available online: 30 March
2017. DOI: http://dx.doi.org/10.17807/orbital.v9i1.897
Abstract: Although phenolic compounds produced by lichens have been widely investigated in antitumor
assays, only a small number have been evaluated for mutagenicity and genotoxicity. This study evaluated
protocetraric, hypostictic, psoromic, and salazinic acids for their potential genotoxic or antigenotoxic activity
against somatic cells of Drosophila melanogaster. These compounds were isolated from the lichens
Parmotrema dilatatum, Pseudoparmelia sphaerosphora, Usnea jamaicensis, and Parmotrema cetratum,
respectively, collected from the Brazilian Cerrado biome. The compounds were evaluated at 0.75, 1.5, 3.0,
and 6.0 mmol L–1 using the SMART test, employing standard and high-bioactivation crosses of Drosophila
melanogaster. Doxorubicin (DXR) was the positive control. Psoromic and salazinic acids proved toxic at 6.0
mM. None of the compounds evaluated exhibited mutagenicity, but each of them significantly reduced
genetic damage caused by DXR, proving antigenotoxic when tested on somatic cells of D. melanogaster.
Keywords: depsidones; genotoxicity; lichens; phenolic compounds; SMART assay
1. INTRODUCTION
Natural products, whether in the form of
extracts, isolated substances, or synthetic and semisynthetic compounds, have been evaluated for their
utility in medicine, the food industry, and agriculture
(e.g., as pest control agents). Their sources are not
limited to higher plants, but include mosses, fungi,
algae, and lichens—all of them producing an
abundance of bioactive substances. In lichens, the
acetate–polymalonate
route
yields
phenolic
compounds
(depsides,
depsidones,
quinones,
anthraquinones, xanthones, dibenzofurans, usnic
acids, and other products). Many of these compounds
have been evaluated for their activity as
antimicrobials, against a wide range of bacteria and
fungi; as antitumor agents, inhibiting growth in a
large panel of tumor cells; as antivirals, inhibiting
replication of viruses, including HIV; and as
inhibitors of enzymes such as 5-lipoxygenase, protein
tyrosine phosphatase, α-glucosidase, and aldose
reductase; among many other activities investigated
[1-3].
*Corresponding
author. E-mail: anamicheletti@gmail.com
Despite the marked activity exhibited by many
lichen compounds, few have been evaluated for
mutagenic or genotoxic properties, crucial for their
safe use as drugs or in other applications. In an early
investigation, Shibamoto and Wei [4] evaluated the
mutagenicity of usnic, physodalic, and physodic
acids. More recently, usnic, diffractaic, olivetoric, and
psoromic acids have been investigated for their
mutagenic and genotoxic potential [5-10].
The wing somatic mutation and recombination
test (SMART) using Drosophila melanogaster was
developed to detect loss of heterozygosity in suitable
gene markers that express detectable phenotypes in
wing cells. Rapid and inexpensive, the method
quantifies, in an unambiguous and highly
reproducible manner, the recombinogenic and
mutagenic potential of chemical and physical agents
[11, 12]. Two crosses—namely, standard (ST) and
high-bioactivation (HB)—are typically used [13]. The
ST cross, obtained from strains expressing basal
levels of the metabolizing cytochrome P450 enzyme
Cyp6A2, is employed to detect direct-acting
genotoxins. The HB cross, obtained from strains
Guterres et al.
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expressing high levels of Cyp6A2, is used to detect
indirect-acting genotoxins that exert their genotoxic
activity only when metabolized [13, 14].
The present study employed the SMART
assay to evaluate the genotoxic and antigenotoxic
activities of protocetraric, hypostictic, psoromic, and
salazinic acids.
2. MATERIAL AND METHODS
General experimental procedures
TLC was performed on pre-coated silica gel 60
GF254 plates (0.20 mm, Macherey-Nagel) and the
spots were visualized by spraying the plates with a
10% sulfuric acid/methanol solution, followed by
heating. Nuclear magnetic resonance (NMR) spectra
were taken on a Bruker DPX-300 spectrometer using
the solvent as an internal reference. Melting points
were recorded on a Uniscience do Brasil 498
apparatus.
Plant collection and extract preparation
Parmotrema
dilatatum
(Vain.)
Hale
(Parmeliaceae), Parmotrema cetratum (Ach.) Hale
(Parmeliaceae) and Pseudoparmelia sphaerospora
(Nyl.) Hale (Parmeliaceae) were collected near
Piraputanga village, in Aquidauana county, Mato
Grosso do Sul state, Brazil (20°27′21.2″S,
55°29′00.9″W; alt. approx. 200 m). Usnea
jamaicensis Ach. (Parmeliaceae) was obtained from
decor stores. Species identification was carried out by
Prof. Mariana Fleig, of the Universidade Federal do
Rio Grande do Sul, Prof. Marcelo P. Marcelli, of the
Instituto de Botânica de São Paulo, and Philippe
Clerc, of the Herbarium of Geneva, Switzerland.
Voucher specimens were deposited at the Campo
Grande Herbarium of the Universidade Federal de
Mato Grosso do Sul (CGMS 49840 for P. dilatatum,
CGMS 37950 for P. cetratum, CGMS 49837 for P.
sphaerospora, CGMS 49838 for U. jamaicensis).
Thalli of P. dilatatum, P. cetratum, P.
sphaerospora, and U. jamaicensis were separately
powdered and extracted with chloroform (2×),
followed by acetone (3×), at room temperature, and
subsequently concentrated in vacuo. The concentrated
acetone extracts were then treated with a small
volume of acetone in an ice bath and centrifuged. This
procedure was repeated until a purified compound
was obtained from each lichen. Protocetraric acid was
51
obtained from P. dilatatum, hypostictic acid from P.
sphaerospora, psoromic acid from U. jamaicensis,
and salazinic acid from P. cetratum. The structures of
these compounds were confirmed by NMR spectra
(Figures S1-S9, Supplementary Material) and were
concordant with the literature [15-17].
Genotoxic activity: somatic
recombination test (SMART)
mutation
and
The SMART assay with D. melanogaster was
performed according to the methodology described by
Fernandes et al. [18]. Three strains were used for
cross breeding: (1) the “multiple wing hairs” (“mwh”)
strain, of mwh/mwh genetic constitution; (2) the
“flare-3” strain, of flr3/In(3LR)TM3 rippsep
l(3)89Aabx34e and BdS genetic constitution; and (3) the
“ORR; flare-3” strain, of ORR/ORR; flr3/In
(3LR)TM3, rippsep l(3)89Aabx34e and BdS genetic
constitution. This last strain inherits chromosomes 1
and 2 from the Oregon R (R) line (which is DDT
resistant), carrying genes responsible for a high level
of metabolizing enzymes of P(CYP)6 A2–type
cytochrome [13]. Two crossings were performed
between these strains: (a) the standard (ST) cross,
from “mwh” males and “flare-3” virgin females [19],
and (b) the HB cross, from “mwh” males and “ORR;
flare-3” virgin females [13].
Eggs from both crossings were collected over
8 h in culture flasks containing a solid agar-agar base
(4% w/v) covered with a layer of biological yeast
supplemented with sugar. Groups of third-instar (72 ±
4 h) larvae were transferred to glass vials containing
alternative medium (1.5 g of instant mashed potato
flakes, Yoki, Brazil) and assayed following two
protocols: (1) for genotoxicity evaluation, each
compound was separately tested at concentrations of
0.75, 1.5, 3.0, and 6.0 mmol L–1; (2) for
antigenotoxicity evaluation, the same concentrations
were employed in association with 2.0 mmol L–1
doxorubicin (DXR). For both protocols, DXR (2.0
mmol L–1) and solvent (Milli-Q water, 1% Tween-80,
and 3% ethanol) were used as the positive and
negative controls, respectively.
Emerging adults carrying one of two
genotypes—namely, marker trans-heterozygous (MH;
mwh +/+flr3) or balancer-heterozygous (BH; mwh+/+
TM3, BdS)—were collected and fixed in 70% ethanol.
The wings were mounted on slides in Faure’s solution
(30 g of gum arabic, 50 g of chloral hydrate, 20 mL of
glycerol, and 50 mL of water) and examined for the
Orbital: Electron. J. Chem. 9 (1): 50-54, 2017
Guterres et al.
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occurrence of mutant spots using an optical
microscope at 400× magnification.
The chi-squared test was employed to interpret
the toxicity assay. Results were considered
statistically significant when p < 0.05.
concentration, however, only psoromic and salazinic
acids proved toxic, significantly reducing survival
rates in treated animals (p ≤ 0.05), compared with the
negative control, which yielded negative or
inconclusive results at this concentration.
CH3 O
CH3 O
Statistical analysis
O
For each treatment, the frequencies of each
type of spot (single small, single large, or twin) and
the total frequency of spots per fly, for each treatment,
were compared in pairs (negative control versus
compounds; DXR alone versus compounds + DXR),
in accordance with the multiple-decision procedure
proposed by Frei and Würgler [20], allowing four
possible diagnoses: positive, negative, inconclusive,
or weakly positive. The relative frequencies of each
group were compared using Kastenbaum and
Bowman’s conditional binomial test [21] at a
significance level of 5%. However, since false
positive results can occur, all final weakly positive
results were analyzed with the non-parametric U-test
[22].
For each compound, inhibition percentages
were calculated from the control-corrected frequency
of clones per 105 cells (FC) and the frequency of
mutation (FM), as follows:
FC = {(DXR alone) – [(DXR alone) – (compound +
DXR)]/(DXR alone)} × 100 [23];
FM = (FC in BH individuals) / (FC in MH
individuals).
The recombination
calculated as FR = 1 – FM.
frequency
(FR)
was
3. RESULTS AND DISCUSSION
The SMART assay was performed to evaluate
the genotoxic activities of protocetraric, hypostictic,
psoromic, and salazinic acids (Figure 1) on the
offspring of ST and HB crosses of D. melanogaster
chronically treated with one of these compounds at
0.75, 1.5, 3.0, and 6.0 mmol L–1.
None of the compounds proved genotoxic,
with frequencies of clone formation per cell division
ranging from 0.41 × 10–5 to 2.15 × 10–5 for the ST
cross and from 0.72 × 10–5 to 3.07 × 10–5 for the HB
cross, therefore not differing significantly from
negative controls (1.6 × 10–5 for ST and 2.25 × 10–5
for HB crosses) (Figure 2). At the highest
52
CH2OH
OH
HO
O
OH
H3CO
CHO
CH3
O
CH3
COOH
HO
1
O
O
2
CH3 O
CH3 O
O
HO
CH3
O
CH3
OCH3
O
CHO
CH2OH
O
OH
HO
O
CHO
COOH
HO
3
O
O
4
Figure 1. Structures of protocetraric (1), hypostictic
(2), psoromic (3), and salazinic (4) acids.
Similar genotoxicity levels for ST and HB
crosses indicate that the enzyme system involved in
cellular detoxification via cytochrome P450 does not
interfere with the genotoxic effect of compounds on
somatic cells of D. melanogaster [24].
The compounds were evaluated not only for
their ability to prevent or induce damage in genetic
material when employed per se, but also for their
ability to prevent DNA damage when administered in
association
with
DXR—an
antineoplastic
anthracycline antibiotic that damages DNA by
interacting with cytosine and guanine, leading to
formation of DNA adducts, which may cause sister
chromatid exchanges, chromosome aberrations, and
interaction with topoisomerase II, preventing
religation of double strands, with permanent DNA
damage
and
subsequent
non-homologous
recombination events [25]. In addition, DXR
generates radicals and oxidative stress, facilitating
lipid peroxidation and ultimately inflicting oxidative
damage to DNA [26].
Figure 3 shows the frequencies of clone
formation in the progeny of ST and HB crosses
treated with non-toxic 0.75-3.0 mmol L–1
concentrations of one of the acids in association with
0.2 mmol L–1 DXR (Tables S1-S8, Supplementary
Material). Again, a consistent pattern was observed
for all four acids, with 72-100% (mostly >80%)
inhibition of mutation events caused by DXR in
Orbital: Electron. J. Chem. 9 (1): 50-54, 2017
Guterres et al.
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descendants of ST and HB crosses. Hypostictic acid
inhibited mutagenic events by 100% both in the ST
cross, when employed at 3.0 mmol L–1, and the HB
cross, when used at 1.5 mmol L–1 and higher
concentrations. Despite their antimutagenic activity,
none of the compounds evaluated had significant
influence on DXR-induced recombination (Tables S1S8, Supplementary Material). As revealed in previous
studies using the SMART assay, the principal
mutational contribution of DXR was related to its
ability to induce recombination.
Figure 2. Control-corrected clone induction frequencies for compounds in the SMART test (NC: negative
control).
Figure 3. Inhibition of mutation events by compounds tested in association with 0.2 mmol L–1 DXR.
DNA changes caused by chemical compounds
can trigger a complex carcinogenesis process. In
normal cells carrying mutations in malignant genes,
loss of heterozygosity by mitotic recombination may
unchain a neoplastic mechanism. Loss of a functional
copy of a heterozygous tumor suppressor gene
53
represents an important step during neoplastic
transformation [27]. Furthermore, mutations that
inactivate tumor suppressor genes or alter expression
of oncogenes may cause malignant transformation
[28]. The compounds evaluated exhibited noteworthy
biological activities and elucidating their mutagenic
Orbital: Electron. J. Chem. 9 (1): 50-54, 2017
Guterres et al.
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profiles paves the way for their future use as
protective agents against mutagenic events.
4. CONCLUSION
Protocetraric, hypostictic, psoromic, and
salazinic acids isolated from Brazilian lichens
exhibited antigenotoxic activity when tested on D.
melanogaster cells, significantly reducing genetic
damage caused by DXR. The antibiotic and antitumor
activities of these compounds lend them for use in
pharmaceutical applications, considering the proven
safety of these substances (absence of DNA damage).
5. ACKNOWLEDMENTS
The authors wish to express their thanks to the
Fundação de Apoio ao Desenvolvimento do Ensino,
Ciência e Tecnologia do Estado de Mato Grosso do
Sul (FUNDECT-MS, Brazil) for its financial support.
Thanks are also extended to Prof. Marcelo P. Marcelli
(Instituto de Botânica de São Paulo, Brazil), Prof.
Mariana Fleig (Universidade Federal do Rio Grande
do Sul, Brazil), and Philippe Clerc (Herbarium of
Geneva, Switzerland) for the identification of lichens,
and to Prof. Adriano A. Spielmann for his support in
the registration of exsiccatae at the Campo Grande
Herbarium of the Universidade Federal de Mato
Grosso do Sul.
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