Ecotoxicology and Environmental Safety 116 (2015) 84–89

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Antioxidant enzymes are induced by phenol in the marine microalga

Lingulodinium polyedrum
P.L.G. Martins a,b,n, L.G. Marques a, P. Colepicolo a
a
Laboratório de Bioquímica e Biologia Molecular de Algas. Departamento de Bioquímica-Instituto de Química-Universidade de São Paulo Av. Prof. Lineu
Prestes, 748-0970 São Paulo, SP, Brazil
b
Centro de Capacitação e Pesquisa em Meio Ambiente (CEPEMA-USP), Universidade de São Paulo. Rd. Cônego Domênico Rangoni, km 271, Cubatão, SP, Brazil

art ic l e i nf o a b s t r a c t

Article history: Knowing the impacts of different anthropogenic activities on ecosystems promotes preservation of
Received 6 November 2014 aquatic organisms. Aiming to facilitate the identification of polluted or contaminated areas, the study of
Received in revised form microalga Lingulodinium polyedrum in phenol-containing medium comprises the determination of toxic
1 March 2015
and metabolic phenol effects, featuring a possible use of this microorganism as bioindicator for this
Accepted 3 March 2015
pollutant. Marine microalga L. polyedrum exposure to phenol increases superoxide dismutase (SOD) and
catalase (CAT) activities. The 20% and 50% inhibitory concentrations (IC20 and IC50) of cells exposed to
Keywords: phenol were 40 μmol L
1 and 120 μmol L
1, respectively. Phenol biodegradation by L. polyedrum was
Environmental toxicology 0.02 μmol h
1 cell
1, and its biotransformation was catalyzed by glutathione S-transferase (GST), phenol
Organic pollutant
hydroxylase and catechol 2,3-dihydroxygenase metabolic pathways. Phenol exposure produced the
Phenol biotransformation
metabolites 2-hydroxymuconic semialdehyde acid, 1,2-dihydroxybenzene (catechol), and 2-oxo-4-pen-
Enzymatic activity
tenoic acid; also, it induced the activity of key antioxidant biomarker enzymes SOD and CAT by three
folds compared to that in the controls. Further, phenol decreased the glutathione/oxidized glutathione
ratio (GSH/GSSG), highlighting the effective glutathione oxidation in L. polyedrum. Overall, our results
suggest that phenol alters microalga growth conditions and microalgae are sensitive bioindicators to
pollution by phenol in marine environments.
& 2015 Elsevier Inc. All rights reserved.

1. Introduction (Doney, 2010; El-Shahawi et al., 2010). Organic compounds that

Diverse human actions that affect the environment are con-
stantly increasing, in turn boosting the demand for measures to
control marine environmental contamination (Cole et al., 2011;
Depledge and Billinghurst, 1999; Halden, 2015; Johnston and Ro-
berts, 2009).
Marine environment contamination occurs when nutrients,
pathogenic toxins, and chemicals are present in levels above en-
dogenous concentrations and/or cause adverse aquatic effects
(Roberts et al., 2008). Toxic contaminants include agricultural and
industrial chemicals such as organic metals, polymers, pesticides,
herbicides, and contaminants generated from domestic waste
(sewage and other debris) (Alzieu, 1991). Organic compounds are
common oceanic pollutants and a public concern, since there are
numerous emission sources, such as industries or agriculture
n
Corresponding author at: Laboratório de Bioquímica e Biologia Molecular de
Algas. Departamento de Bioquímica-Instituto de Química-Universidade de São
Paulo Av. Prof. Lineu Prestes, 748-0970 São Paulo, SP, Brazil.
E-mail addresses: paula@cepema.usp.br, paula.sfc@gmail.com (P.L.G. Martins).
affect coastal areas include organochlorines, pesticides, petroleum
derivatives and phenolics (Dsikowitzky et al., 2011).
Phenol is a chemical compound that results from the de-
gradation and oxidation of aromatic organic compounds including
benzene, polymers and others (Enache et al., 2010; Zagatto and
Bertoletti, 2008). Phenol is a byproduct of many industrial pro-
cesses involving coal gasification, coke production, pharmaceu-
ticals, pesticides, fertilizers, refineries, steel, ceramics, phenolic
resins, paints, synthetic chemistry, paper, and pulp (Michalowicz
and Duda, 2007). Pollutants generated from these industrial pro-
cesses, including phenol and many others, are currently under
major consideration by environmental protection agencies
worldwide.
The utilization of marine organisms in biomonitoring is of great
relevance to identify the effect of a contaminant at low con-
centrations, the worldwide oceanic impact, and to determine ac-
tions needed to contain and recover a contaminated area. Aquatic
organism (or biomonitor) toxicity refers to the toxic effect physical
and chemical agents exert on an organism that is representative of
an aquatic environment (Tatken and Lewis, 1983).

http://dx.doi.org/10.1016/j.ecoenv.2015.03.003
0147-6513/& 2015 Elsevier Inc. All rights reserved.
 P.L.G. Martins et al. / Ecotoxicology and Environmental Safety 116 (2015) 84–89
Microorganisms are the most popular bioindicators. The bio-
monitoring capabilities of microalga Lingulodinium polyedrum,
such as its antioxidant capacity, cellular behavior of acclimatiza-
tion, photosynthetic capacity, biodegradation properties, and
ability to cope with chemical toxicity in preliminary ecotoxicity
studies, are described (Ganini et al., 2013). Studies with L. poly-
edrum explored physiological processes related to its biological
clock, such as bioluminescence, cell division, vertical migration,
photosynthesis, nitrate reductase and superoxide dismutase (SOD)
activities (Cardozo et al., 2002). Leitão et al. (2003) showed that
polychlorinated biphenyl (PCB) exposure significantly inhibits
cellular antioxidant defenses and acclimation in L. polyedrum, and
these results formed the basis for our study. The exposure of mi-
croalgae to phenol was also explored by Semple and Cain (1996)
and Campos (2008), who described the biotransformation of
phenol by the microalgae Ochromonas danica and Minutocellus
polymorphus, respectively. We believe L. polyedrum is part of an
important cellular group, that should be investigated for metabolic
reactions and toxic effects caused by exposure to phenolic com-
pounds (Lika, 2009).
High occurrence of L. polyedrum in the aquatic environment,
distributed over the northern hemisphere in colder waters of the
Atlantic Ocean, assures a interesting bioindicator potential (Dale,
1996). L. polyedrum was also chosen because of the need for a
marine microorganism biomonitor in standardized toxicity tests,
and because many of its biochemical pathways have already been
characterized. Furthermore, literature (Torres et al., 2008) sup-
ports its use in biochemical assays monitoring exposure and sen-
sitivity exposure to a variety of environmental contaminants.
The ecotoxicological analyses performed in this study provided
important information about phenol-exposed L. polyedrum bio-
chemistry, a biomonitor that can be used to report marine biota
toxicity. The objective of this study was to examine bio-
transformation processes and antioxidant activity in the microalga
L. polyedrum after phenol exposure, aimed to identify and isolates
toxicity in marine ecosystems.
2. Materials and methods
2.1. Cell culture
L. polyedrum cells were cultured in Guillard F/2 medium
(Guillard and Ryther, 1962) at 20 °C with alternating 12 h white
illumination in a Tecnals TE-4001 growth chamber. The light in-
tensity was 50 mmol m
2 s
1.
2.2. Phenol quantification and biotransformation
Culture samples (1 mL) were collected and purified (i.e., algae
content was removed) with 0.22 μm Millipore filters. High-per-
formance liquid chromatography (HPLC) was performed to mea-
sure phenol concentrations as previously described by Thakur
et al. (2002). The equipment used was a Shimadzu UFLC with
autosampler, C18 column (Phenomenexs, 30  2.1 mm2, 5 mm)
and UV–vis detector at a wavelength of 254 nm. The solvent sys-
tem was acetonitrile (pump A) and H2O þ0.2% formic acid (pump
B), at 0.9 mL min
1, a total time of 17 min run and gradient going
from 60% to 20% of pump B between 3 and 15 min. Controls
(phenol in the culture medium without cells) were used to con-
firm the presence of phenol in the experiments. The limit of de-
tection found for phenol was 0.009 mmol L
1 and the limit of
quantitation was 0.05 mmol L
1. To identify metabolites, stan-
dards were prepared with phenol and catechol (Mercks) and in-
jected into a gas chromatography-mass spectrometry (GC–MS)
instrument using chloroform (Vetecs) as solvent. The stock phenol
85
solution (10 mmol L
1 in chloroform) was diluted to 6 points of
concentrations 0.05–1.0 mmol L
1 for calibration curve construc-
tion. (Silvestre et al., 2009). Aqueous organic metabolites were
extracted using a proportion of 2:1 chloroform:sample (v/v), vor-
texed for 2 min, and the organic phase was recovered. Residual
water was removed with anhydrous sodium sulfate (Vetecs) and
samples were suspended in chloroform and injected in the GC–MS
system.
Metabolites were separated and measured by GC–MS in ne-
gative ionization mode (GC–MS Variums) using an SPB-20 column
(Sigma-Aldrichs) for a total run time of 26 min, using these
temperature conditions: injection at 250 °C; initial temperature of
50 °C oven for 5 min; then, addition of 1075 °C per min up to
280 °C. Biotransformation fragments were identified with the
National Institute of Standards and Technology (NIST) Bibliotheca.
2.3. Toxicity assays
Dose-response (DR) curves were performed to calculate the
phenol concentration required to inhibit cell growth by 20% (IC20)
and by 50% (IC50) in L. polyedrum cultures (U.S. EPA, 1971, 2007).
Phenol (0.05–1.0 mmol L
1 or 4.8–94 mg L
1) was added to cul-
tures with a density of 4  103 cells mL
1. After 24 h, cell viability
was determined by optical microscopy (Heasman et al., 2000) with
Evan's Blue dye (Sigma-Aldrich), a dye that leaks through damaged
cell walls. Unhealthy cells were not counted, as phenol toxic effects
include induction of cystic cells stage and/or damage to cell wall.
DR curves were performed in triplicate. Controls were done using
untreated cells and cell-free medium. Phenol concentration in
culture medium was measured by HPLC-UV, as described in Sec-
tion 2.2. The software used to perform these calculations was IC-
PIN.EXE using Linear Interpolation Method.
2.4. Antioxidant enzyme activity
Standardized enzyme activity assays were performed by
quantifying the total protein in the samples with Bradford assay.
The enzymatic activity of superoxide dismutase (SOD) was mea-
sured according to the method described in McCord and Fridovich
(1968). Catalase (CAT) activity was performed according to Aebi
(1984) by spectrophotometrically monitoring the decomposition
of H2O2. Ascorbate peroxidase (APX) was issued according to
Asada (1984) by monitoring the oxidation of ascorbate in the
presence of H2O2. Glutathione S-transferase (GST) activity de-
termination was provided for monitoring 1-chloro-2,4-dini-
trobenzene in presence of GSH, described in Pflugmacher et al.
(2000). All enzymatic activity assays were measured in control
cultures and in cultures treated with phenol for 24 h.
The intracellular GSH/GSSG ratio (Romano, 2009) was mea-
sured with an Applied Biosystemss 3200 QTrap liquid chroma-
tography-mass spectrometry (LC-MS) system. C18 column was
used (Luna, 250  4,6 mm2, 5 mm, Phenomenexs), with two mo-
bile phases as the solvent system: water with 5 mmol L
1 am-
monium acetate and 0.1% formic acid (pump A) and methanol/
water (% 65/35 v/v) containing 5 mmol L
1 ammonium acetate
and 0.1% formic acid (pump B), at 0.9 mL min
1; with gradient
going from 0% to 100% of pump B between 0 and 5 min. The total
run time was 10 min. In order to prevent oxidation of GSH in the
preparation of the standard curve and samples, 100 mL N-ethyl-
maleimide (1 mol L
1) were added as the derivatizing agent,
which ensured the necessary chemical stability. Curves were
prepared using standards from Sigma-Aldrichs and five points,
from 0.05 to 5 μg mL
1 of GSH and GSSG.
86 P.L.G. Martins et al. / Ecotoxicology and Environmental Safety 116 (2015) 84–89
3. Results and discussion
3.1. Toxicity
Dose-response curves generated from phenol (0.05, 0.10, 0.15,
0.25, 0.50 and 1.0 mmol L
1) treated cells allowed us to calculate
IC20 and IC50 values. The determined IC20 was 0.04 mmol L
1, and
IC50 was 0.12 mmol L
1. The 95% confidence interval for IC50 va-
lues were 0.93 and 0.14 mmol L
1 (Table 1). IC50 was used in ex-
periments for metabolite identification. For enzyme activity as-
says, cells were treated slightly below the IC50 (0.10 mmol L
1
phenol) because a greater algal mass was required for assays.
Table 1-IC20 and IC50 for phenol-treated cells.
For marine microalga M. polymorphus, IC20 and IC50 for phenol
were 1.5 and 2.3 mmol L
1, respectively (Walsh et al., 2008). Our
results suggest phenol is less toxic towards M. polymorphus than L.
polyedrum. However, comparisons are difficult because phenol
exposure conditions are not standardized between studies. Eklund
and Kautsky (2003) argue for the creation of standards to compare
IC and effective concentration (EC), and highlight the difficulty
associated with comparing results between studies.
Another study by De Morais et al. (2014) evaluated the cya-
nobacterium Microcystis aeruginosa response to penta-
chlorophenol (more toxic than phenol) in comparison with that of
the microalga Chlorella vulgaris. They observed that M. aeruginosa
could remove part of the pentachlorophenol from the medium, at
concentrations where toxic effects were observed, while C. vulgaris
stabilized it. The toxicity profiles of the two species were very
different. The calculated effective concentrations EC20 and EC50
were much lower to M. aeruginosa, consolidating the needs of
studying the toxicological effects in different species of microalgae
when exposed to innumerable contaminants, in order to generate
a reference database.
3.2. Phenol biotransformation
Chromatography was used to measure phenol in the culture
medium and intracellular material. Phenol consumption was de-
termined by calculating the absorption rate of phenol (Rx) given
by (1)
Rx=(μmol phenol/24)/cells number (1)

1
The microalga L. polyedrum removed 0.12 mmol L of phenol
from the medium 24 h after exposure, with an average removal
rate of 0.02 μmol h
1 cell
1.
To make a comparison, it can be said that M. polymorphus was
able to remove 250 μmol L
1 of phenol from the culture medium
in 6 days of exposure; also, cells previously exposed to phenol
removed 99% of this pollutant after 2 h (Walsh et al., 2008). Given
that phenol concentrations that inhibit growth are distinct be-
tween species and chronic exposure studies are lacking, it is dif-
ficult to predict growth characteristics of different microalgae
under identical test conditions. However, our results suggest that
M. polymorphus has a greater phenol biotransformation capacity
than L. polyedrum.
Table 2 includes the m/z ratio of molecular ions identified in
Table 1
Values IC 20 and IC 50 of cells treated with phenol.
Cells concentration Phenol concentration
IC 20 ( E3,8  10³ cells mL
1) 0,04 mmol L
1 or 40 lmol L
1
IC 50 ( E2,4  10³ cells mL
1) 0,12 mmol L
1 or 120 lmol L
1
IC 20¼ concentrations of growth inhibition of 20%
IC 50¼ concentrations of growth inhibition of 50%
cell extracts analyzed by GC–MS. Table 2 lists compounds identi-
fied from chromatogram peaks and their corresponding mass
spectra. Several peaks were identified as corresponding to three
phenol metabolites.
Table 2-Metabolism: Molecular weights identified by GC–MS.
Chromatogram peaks corresponded to the acid metabolites
hydroxymuconic-2-semialdehyde, 1,2-dihydroxybenzene (ca-
techol) and 2-oxo acid 4-pentenoic acid (Tsirogianni et al., 2005).
There were also unidentified peaks, most likely due to various
cellular components or ionization source byproducts.
The identified fragments suggest a phenol metabolic pathway
(Fig. 1) identical to the one existing in previously studied micro-
algae, including Minutocellus polymorphus (Walsh et al., 2008),
Chlorella sp. (Tikoo et al., 1997), and Ochromonas danica (Semple
and Cain, 1996; Semple et al., 1999). The presence of the acid
metabolite hydroxymuconic-2-semialdehyde suggests catechol
2,3-dioxygenase enzyme activity in L. polyedrum. The presence of
catechol’s acid metabolite hydroxymuconic-2-semialdehyde in
phenol-treated cells, further, suggest increased catechol 2,3-diox-
ygenase enzyme activity.
3.3. Enzyme assays — antioxidant and phase II biotransformation
The SOD activity was increased by 3-fold (98 U mg
1 protein)
in treated samples when compared to controls (Fig. 2).
CAT activity was also increased (10.54 μmol min
1 mg
1) in
the treated cells compared to controls (8.39 μmol min
1 mg
1)
(Fig. 2).
CAT is an antioxidant complex that metabolizes H2O2 produced
by ROS. Increased CAT activity in L. polyedrum suggests excess of
H2O2 in treated cells. Increased CAT activity repairs the cell and
prevents massive accumulation of ROS (Pinto et al., 2000).
SOD and CAT activity may be early biomarkers of antioxidant
activity in response to phenol exposure in L. polyedrum cells. Xe-
nobiotic phenolic derivative biotransformation that generate ROS
(Goeptar et al., 1995) can uncouple cytochrome P450 reactions,
produce O2
and H2O2, and further promote oxidative damage in
microalgal biological systems (Ganini, 2012). The generation of
ROS may also be associated with changes in the antioxidant de-
fenses within various aquatic organisms (Livingstone et al., 1991).
The activities of antioxidant enzymes, SOD and CAT were also
enhanced remarkably at low concentrations of uniconazole (aro-
matic compound used for sowing) on two marine microalgal
species (Mei et al., 2014).
Phenol had the opposite effect on ascorbate peroxidase (APX)
activity than on SOD and CAT. APX activity was increased in con-
trols compared to treated cells, although not significantly, as
shown by a p-value of 0.08 in the ANOVA test performed (Fig. 2).
It is likely that the CAT peroxidase enzyme has increased ac-
tivity due to high H2O2 concentrations induced by phenol. It is our
opinion that L. polyedrum adapted to decrease APX and increase
CAT in response to high H2O2 concentrations in cultures exposed
to phenol.
Data described in Fig. 2 suggest that CAT is the most effective
system for reducing H2O2 in L. polyedrum. APX has a greater affi-
nity for hydrogen peroxidase than CAT; however, APX is located in
chloroplasts, whereas CAT is located in peroxisomes. Therefore,
the location of ROS production requires further investigation.
The red alga Kappaphycus alvarezii presented similar APX and
CAT activities after exposure to the organic compound clofibrate
(Barros et al., 2003).
We determined that phenol induced SOD and CAT antioxidant
activities in the microalga L. polyedrum. Both SOD and CAT were
excellent biomarkers of oxidative stress.
Glutathione S-transferase (GST) activity was increased three-
fold (0.09 mmol min
1 mg
1) in treated microalgae compared to
 P.L.G. Martins et al. / Ecotoxicology and Environmental Safety 116 (2015) 84–89 87

Table 2
Metabolism-molecular weights found in GC–MS.

Molecular structure metabolite m/z precursor ion Potential fragments m/z Fragments ions

MW¼ 142,00 141 MW ¼ 101,02 101

MW ¼ 42,01 41

MW¼ 110,04 110 MW ¼ 84, 02 83

MW¼ 114,03 113 MW ¼ 98,04 97

MW ¼ 72,99 72

MW ¼Molecular Weight

OH OH OH
2H + O 2 H 2O OH O2
COOH
CHO
Phenol hydroxylase Cathecol-2,3-dioxygenase
Fig. 1. Via suggested for the degradation of phenol by L. polyedrum.

controls (0.03 mmol min
1 mg
1) (Fig. 2). described in Fig. 1, elevated GST activity in treated cultures sug-
Our results suggest that GST caused detoxification and rapid gests phenol is also biotransformed by conjugation with
excretion of phenol by cells. In addition to cellular metabolism glutathione.

Fig. 2. Activity of Superoxide dismutase (SOD), Catalase (CAT), Ascorbate peroxidase (APX) and Glutathione S-transferase (GST) in cultures of L. polyedrum after 24 h
exposure to 0.10 mmol L
1 phenol. Values represent triplicates experiments average with standard deviations. n Indicate ANOVA signification, p o 0,05.
88 P.L.G. Martins et al. / Ecotoxicology and Environmental Safety 116 (2015) 84–89
Fig. 3. GSH/GSSG ratio in cultures of L. polyedrum after 24 hours exposure to 0.10
mmol.L-1 phenol. Values represent triplicates experiments average with standard
deviations. n Indicate ANOVA signification, po 0,05.
GST also biotransforms phenol in M. polymorphus. However,
GST activity (7.8 and 14.0 nmol min
1 mg
1 in the treated and
control groups, respectively) was lower in M. polymorphus than in
L. polyedrum exposed to phenol.
The GSH/GSSG ratio in treated samples was lower than that in
controls, especially because of an increase of 20 ng  mL-1 GSSG in
treated microalgae ( Fig. 3). Elevated GST activity in treated groups
suggests efficient GSH turnover and biosynthesis by this micro-
alga. Furthermore, GSH levels remained slightly below control le-
vels. GSH aids rapid excretion of phenol from cells, and in treated
groups, our results suggest that GSH was maintained at sufficient
levels for GST detoxification of phenol in L. polyedrum.
Therefore, our results suggest that the biotransformation of
phenol in L. polyedrum occurs by GST catalyzed conjugation with
glutathione, and that phenol is further detoxified by the hydro-
xylase and catechol-2, 3-dioxygenase metabolic pathways.
Our results indicated that phenol exposure altered microalgae
antioxidant and metabolic systems and suggest that L. polyedrum
is a relevant bioindicator to marine pollutants and contaminants,
especially phenol – and possibly to compounds with chemical
characteristics similar to it.
4. Conclusion
The organic compound phenol increased the antioxidant ac-
tivity in the microalga L. polyedrum. The enzymes SOD and CAT
(induced three-fold in phenol-treated microalgae) were identified
as biomarkers of increased antioxidant activity. We expect future
marine toxicity tests for potential aromatic compound con-
tamination to use L. polyedrum as a biomonitor and assay for the
biomarkers SOD and CAT. Furthermore, biotransformation of
phenol in L. polyedrum is performed by GST-catalyzed conjugation
with glutathione, and phenol is further detoxified by hydroxylase
and catechol 2,3-dioxygenase metabolic pathways.
Acknowledgments
CEPEMA-USP: Center for Environmental Training and Research,
University of São Paulo, FUSP and all that equipped financial and
physical support.
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