Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

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Comparative Biochemistry and Physiology, Part C

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Molecular and biochemical effects of the antifouling DCOIT in the mussel T

Perna perna
⁎
Heloísa Bárbara Gabe, Amanda da Silveira Guerreiro, Juliana Zomer Sandrini
Programa de Pós-Graduação em Ciências Fisiológicas. Instituto de Ciências Biológicas, ICB. Universidade Federal do Rio Grande - FURG, 96203-900 Rio Grande, RS,
Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Biological fouling is an unwanted phenomenon that results in economic losses to the shipping industry. To
Mollusk prevent fouling, antifouling paints are used. DCOIT (4,5- dichloro-2-n-octyl-4-isothiazolin-3-one) is a biocide
Oxidative stress present in many antifouling paint formulations, and is toxic to a wide range of organisms. The aim of the present
Biocide study was to evaluate the effects of DCOIT on oxidative stress indicators of the brown mussel, Perna perna.
Biomarker
Molecular (SOD-like, GSTO-like and MGST-like mRNA levels) and biochemical (activities of superoxide dismutase
Antioxidant defence
(SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH), reactive oxygen
species (ROS) and protein carbonyls (PCO)) components were evaluated. Further, levels of biomarkers were
assessed in the gills and digestive glands of mussels. Bivalves were exposed to DCOIT (control, 0.1 μg/L and
10 μg/L) for up to 96 h. DCOIT exposure decreased GSH content in gills. Moreover, exposure to DCOIT also
decreased CAT activity in the gills and digestive glands of mussels. GST activity increased in digestive gland after
exposure for 24 h to both concentrations of DCOIT tested. SOD activity, ROS levels and PCO content were not
affected by exposure to the contaminant. Regarding the molecular biomarkers evaluated, DCOIT exposure al-
tered mRNA levels of SOD-like in both tissues after 24 and 96 h of exposure, and decreased MGST-like mRNA
levels in the digestive gland after 96 h of exposure to the chemical. These findings suggested that exposure to
DCOIT may alter the biochemical and molecular functioning of P. perna, which may harm the species.

1. Introduction et al., 2000; Martínez et al., 2001). No information regarding the pre-
sence or levels of DCOIT throughout the Brazilian coast has been re-
Biofouling is characterized as the accumulation of animals, plants, ported in literature.
algae and bacteria in submerged structures (Yebra et al., 2004). This Although many studies have reported its presence in marine waters,
process is considered an unwanted phenomenon which can result in DCOIT is a biocide considered environmentally acceptable as anti-
economic losses for the shipping industry (Champ, 2000). To prevent fouling agent due to its rapid degradation in the marine environment
the attachment of fouling organisms, antifouling paints have been used. (Chen and Lam, 2017). Biological degradation of DCOIT by micro-
However, some biocides present within paints are toxic to non-target organisms is considered to be over 200 times faster than hydrolysis or
organisms (Amara et al., 2018) and can be highly persistent in the photolysis (Willigham and Jacobson, 1996, Jacobson and Willingham,
environment (Readman, 2006). A global ban on the antifouling biocide, 2000). Jacobson and Willingham (2000) and Sakkas et al. (2002), for
tributyltin (TBT), in 2008 catalyzed the increased use of alternative example, demonstrated that the biocide has a half-life of 1 to 13 d and
antifouling booster biocides, such as DCOIT (4,5-dichloro-2-n-octyl-4- the principal degradation product of the compound is N-(octyl)car-
isothiazolin-3-one), a biocidal ingredient of Sea Nine 211™. bamic acid (Thomas, 2001).
Due to its exclusive use as a component of antifouling paints, DCOIT DCOIT is a member of the isothiazolone group of compounds. It can
is generally associated with harbors, docks and platform structures. rapidly diffuse though cell membranes and is highly toxic to organisms
Environmental concentrations of DCOIT in marina areas in Japan (Collier et al., 1990). According to Arning et al. (2008), DCOIT can also
(Harino et al., 2006), Greece (Sakkas et al., 2002), Denmark (Steen interact with the tri-peptide glutathione (GSH), which is an important
et al., 2004) and Spain (Martinéz et al., 2000) have been reported to component of the defence response of organisms. The combination of
range from 0.1 μg/L (Tsunemasa et al., 2006) to 3.7 μg/L (Martinéz the lipophilicity and GSH reactivity of DCOIT may damage the

⁎
Corresponding author.
E-mail address: juzomer@pq.cnpq.br (J.Z. Sandrini).

https://doi.org/10.1016/j.cbpc.2020.108870
Received 8 February 2020; Received in revised form 28 July 2020; Accepted 9 August 2020
Available online 16 August 2020
1532-0456/ © 2020 Published by Elsevier Inc.
H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

antioxidant defence systems of organisms as well as their bio- dissected. Tissues (gills and digestive gland) were separated and stored
transformation processes (Arning et al., 2008). Other studies have de- at −80 °C for further analysis.
monstrated that the mechanistic details of DCOIT toxicity may also
involve the formation of free radicals (Chapman and Diehl, 1995). 2.3. Molecular analysis
Blockage of the antioxidant systems of organisms, which occurs through
GSH impairment and ROS generation, may produce oxidative stress, Total RNA extraction was performed in gills and digestive glands of
and therefore, oxidative damage (Chen et al., 2014a). mussels using TRI reagent (Sigma-Aldrich, St. Louis, USA) according to
Studies evaluating DCOIT toxicity have demonstrated that it can be the manufacturer's instructions. RNA purity was determined spectro-
responsible for the perturbation of the mitochondrial respiratory chain photometrically using a BioDrop μLite spectrophotometer by measuring
in hemocytes of ascidian Botryllus scholosseri (Cima et al., 2008). Fur- the absorbance ratio at 260/280 nm. Residual genomic DNA was re-
ther, the compound has been shown to inhibit ATP synthesis in rat liver moved by treating with DNase I (Sigma-Aldrich, St. Louis, USA).
mitochondria (Bragadin et al., 2005). For polychaetes like Perinereis Synthesis of cDNA was performed using 1 μg of total RNA per sample.
aibuhitensis and marine mysids like Neomysis awatschensis, DCOIT ex- Briefly, reverse transcription was performed using a mixture of Oligo-
posure has been reported to induce changes in antioxidant defence dT and random primers (100 μM), RNase inhibitor (20 U), 500 nM
systems (Eom et al., 2019) and negatively affect the growth of animals dNTP and 400 U M-MLV Reverse Transcriptase (Sigma-Aldrich, St.
via the inhibition of AChE activity (Do et al., 2018). Other studies have Louis, USA).
reported that DCOIT is toxic for bivalves, and Bellas (2006) demon- Real-Time PCR were performed with an Applied Biosystems 7300
strated that the biocide is toxic to early-developmental-stage Mytilus Real Time PCR System. Real-Time PCR reactions were conducted in
edulis. Further, Tsunemasa and Okamura (2011) showed that the duplicate using GoTaq qPCR Master Mix (Promega, Madison, USA) and
compound strongly impacted oyster development. diluted cDNA (1:10) was used as a template for the amplification
Bivalve mollusks are particularly useful bioindicator species (Bayne, fragments of genes listed in Table 1. The PCR amplification reaction
1978; Savorelli et al., 2017; Faggio et al., 2018) since they filter-feed parameters included an initial 2 min step at 95 °C, followed by 40 cycles
and are semi- sessile. Due to the large demographic distribution of bi- at 95 °C for 15 s, 60 °C for 15 s and 60 °C for 30 s, and a final elongation
valves, which live mainly in coastal regions, and to their filter-feeding step at 60 °C for 30 min. The melt curve was programmed to run from
activities, the animals are particularly vulnerable to anthropogenic 65 °C to 95 °C. PCR efficiency was determined for each primer by
activities (Capilo et al., 2018). Mussels are well known for their sentinel constructing a standard curve from serial dilutions of cDNA. Actin, ef1α
characteristics (Resgalla Jr. et al., 2008) and for their economic im- and tubulin were used as reference genes. To calculate the normalization
portance. In world, production of the mussel reached 23,000,000 tons factor, the geNorm algorithm was used (Vandesompele et al., 2002).
in 2017 (FAO, 2017). However, no studies investigated the adverse eff After converting the CT values into quantities using the standard
ects of DCOIT on P. perna. Given that DCOIT can alter antioxidant de- curves, the levels of gene expression were normalized through the
fence systems and produce an imbalance between these defence and the normalization factors generated by geNorm. Sequences were obtained
production of ROS, the present study examined the effects of the bio- through the transcriptome analysis of Perna perna samples (BioProject:
cide DCOIT in the gills and digestive glands of the brown mussel P. PRJNA531290; Monteiro, 2017) and sequence identification was per-
perna. formed by Guerreiro et al. (2020b).
PCR efficiency was determined for each primer by constructing a
2. Methods standard curve from serial dilutions. Actin, Ef1α and Tubulin were used
as reference genes.
2.1. Animals
2.4. Biochemical analyses
Mussels (Perna perna) were obtained from a mariculture farm lo-
cated in the southwest region of Santa Catarina, Brazil. After collection, 2.4.1. Reactive oxygen species
animals were transported to the laboratory at the Federal University of To analyse the level of ROS generated post-DCOIT exposure, tissues
Rio Grande – FURG and maintained under constant conditions (tem- (gills and digestive glands) were homogenized (1:4 w/v) in cold buffer
perature, 20 °C; salinity, 30‰ and photoperiod of 12 h light/12 h dark), (100 mM Tris-HCl, 2 mM EDTA, 5 mM MgCl2, pH 7.75). Samples were
proportion of 1.5 L seawater for each animal in 60 L plastic buckets, centrifuged at 20,000 xg for 20 min at 4 °C and supernatants were
constant aeration, for approximately 15 d. During this period, animals collected. Supernatants were then diluted to a final protein concentra-
were fed with 10 mL of phytoplankton per bucket including Conticriba tion of 2.2 mg/mL, using the same buffer as was used previously. Total
weissflogii, Nannochloropsis sp., Chaethoceros muelleri and Isochrysis gal- protein levels within samples were determined via the Biuret method,
bana and their water (filtered) was replaced every 2 d. which was facilitated through use of a commercial kit (Doles Reagentes
Ltd., Goiânia, Brazil). The levels of ROS were analysed as described by
2.2. DCOIT exposure Ferreira-Cravo et al. (2009), where the reactive oxygen molecules
present in the sample react with 2′,7′-dichlorodihydrofluorescein dia-
Animals were exposed to two concentrations of DCOIT (0.1 and cetate generating a fluorochrome that is detected fluorometrically with
10 μg/L). To treat animals, DCOIT previously dissolved in di- a 485/520 nm filter for 30 min.
methylsulfoxide (DMSO) was used. The final solvent level of all tanks
used for all experiments was 0.016%. A control group was exposed 2.4.2. Antioxidant defence
exclusively to DMSO (0.016%). DCOIT was purchased from Sigma To evaluate levels of SOD, CAT, GST activities and assess levels of
(Sigma-Aldrich, St. Louis, USA). The 0.1 μg/L concentration was se- non-proteic thiols (glutathione); tissues (gills and digestive gland) were
lected based on observed environmental levels (Tsunemasa et al., 2006) homogenized in cold buffer (20 mM Tris base, 1 mM ethylenediami-
and the 10 μg/L concentration was selected based on its reported netetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 500 mM sucrose,
toxicity against Mytilus edulis larvae (Bellas, 2006). 150 mM KCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF),
Mussels were divided into 9 tanks (4 animals per tank, three tanks pH 7.6) at a ratio of 1:4 (w/v). Homogenates were centrifuged (1000 g
per goup) containing 6 L filled with seawater as presented in Fig. 1. for 20 min at 4 °C and 10,000 g for 45 min at 4 °C) and supernatants
During the experimental period, animals were not fed, and water was were collected. Samples were stored at −80 °C until they were used for
renewed daily. After 24 h and 96 h of exposure, 6 animals per treatment enzymatic assays and the measurement of non-protein thiol (glu-
(n = 6 samples per group) were killed on ice and immediately tathione) levels.

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H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

Fig. 1. Schematic diagram of the experimental design of this study. Mussels placed in 6-L tanks (three for each tratament) were exposed to either 0, 0.1 or 10 μg/L of
DCOIT. After 24 and 96 h of exposure, mussels (n = 6) were dissected, and gills and digestive glands were collected.

Table 1 (1969). Phosphate buffer 50 mM (pH 7.8) is used for reaction. The
Primer sequences used for the amplification target gene fragments via real-time analysis is based on the oxidation of cytochrome C (1 μM), which occurs
PCR. as a result of the generation of superoxide anions by the xanthine/
Primer Sequence (5′ – 3′) Amplicon PCR xanthine oxidase system. The reaction was monitored spectro-
length Efficiency photometrically at 550 nm for 1 min. Results are expressed in units of
SOD, which represent the amount of enzyme (sample) required to in-
SOD-like F: 5′ - CCC TCC CGC GTG AGA ACT CAT T – 125 bp 92.8%
hibit cytochrome C oxidation 50% per min and per mg of protein at
3′
R: 5′ - GCA TGC AAC TCT TCC GCC AGC – 3′
25 °C. The analysis was performed in triplicate.
GSTO-like F: 5’ – AGG TTC CGA GTG TCC GCC ATT – 3′ 191 bp 114% GSH levels were determined as described by Sedlak and Lindsay
R: 5′ – AGT CGT GTT CGT TGA GCG TAT GG (1968). The method is based on protein precipitation and the sub-
– 3′ sequent reaction of non- proteic thiols with 5,5-dithio-bis-(2-ni-
MGST-like F: 5’ – CGG AAT GGT CTG GCT ACT TG – 3′ 87 bp 110%
trobenzoic acid) (DNTB). The reaction can be detected using a fluor-
R: 5‘– GCA AAC GCT CCT CTC ATT CT – 3′
EF1∝ F 5′ - ACC ACG AGT CAA TGC CAG AG – 3′ 106 bp 91.8% escence microplate reader. Thiol groups were evaluated at 415 nm and
R: 5′ - TTG CTG TCA CCA CAG ACG TT – 3′ their concentrations were expressed in nmoles per mg protein.
Tubulin F 5′ – AGC TGA CCC TAA ACG CAG TC – 3′ 198 bp 92.2%
R 5′ – TTG GCT AGC GTC AGG AGA GA – 3′
2.4.3. Protein carbonyl content
Actin F: 5′ – CAG GAT CTG GCG ACA TGG TT – 3′ 139 bp 92.7%
R: 5′ – CAG GCT TGT GGT CCT GAA CT – 3′ Carbonylated protein content was measured using a commercially
available kit according to the manufacturer's instructions (OxiSelect TM
Protein Carbonyl Fluorometric assay; number MBS168032). The pro-
Catalase activity was determined as described by Beutler (1975). tein carbonyl content within protein samples was first derivatised using
Briefly, this assay requires the detection of the degradation of hydrogen a protein carbonyl fluorophore. Proteins were then trichloroacetic acid
peroxide (H2O2, Sigma Aldrich) at 240 nm using a spectrophotometer, solution (TCA) precipitated and free fluorophores were removed by
for 1 min. Buffer was used (Tris Base 1 M, EDTA 5 mM, pH 8) and the washing the protein pellet with acetone. After dissolving the protein
tissue sample is homogenized. Results were expressed in units of CAT, pellet in guanidine hydrochloride, the absorbance of the protein-
which is defined as the amount of enzyme required to hydrolyse 1 μmol fluorophore product was measured fluorometrically using a 485/
of H2O2 per min per mg of protein at 25 °C. 520 nm filter. The results have been expressed in nmol per mg protein.
Glutathione S-transferase (GST) activity was measured based on the
protocol of Habig et al. (1974). Phosphate buffer 0,1 M (pH 7) is used 2.5. Statistical analysis
for reaction. This assay assessed the formation of a GSH (25 mM)
conjugate with CDNB (1-chloro-2,4-dinitrobenzene) (50 mM) which Data are expressed as mean ± standard error. Statistical analysis
was measured by detecting its absorbance for 2 min at 340 nm. Results was performed in Prism software, where a one-way ANOVA followed by
were expressed in units of GST, which represent the amount of enzyme the Tukey post-hoc multiple range test was accessed. All prerequisites
required to conjugate 1 μmol of CDNB per min and per mg of protein at for the analysis of variance (normality and homoscedasticity) were
25 °C. tested previously. A statistical probability of p < 0.05 was considered
The SOD activity was quantified according to McCord and Fridovich significant.

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H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

Fig. 2. mRNA levels (mean ± standard error) of SOD-like (A and B), MGST-like (C and D), and GSTO-like (E and F) in gills (left) and digestive glands (right) of P.
perna exposed to 0.1 μg/L and 10 μg DCOIT. Different letters indicate significant differences between groups indicated.

3. Results unaltered by DCOIT exposure in gill tissue (Fig. 2C), but were reduced
in digestive gland tissue after exposure to 10 μg/L DCOIT (96 h)
No mortality was observed during the experiments. Overall, the (Fig. 2D). On the other hand, GSTO-like mRNA levels were not sig-
exposure of mussels to DCOIT resulted in changes in some parameters nificantly altered (p > 0,05) in either the gills or the digestive glands
of the antioxidant defence system present in both gills and the digestive of P. perna (Fig. 2E,F) when either concentration of DCOIT was tested.
glands of animals.
3.2. Antioxidant defences
3.1. Molecular biomarkers
While the activity of superoxide dismutase (SOD) was not altered
DCOIT exposure induced changes in the mRNA levels of some genes after the either gill (Fig. 3A) or digestive gland (Fig. 3B) tissues were
evaluated (Fig. 2). In the gills, an increase in SOD-like mRNA expression exposed to DCOIT, the enzymatic activity of CAT was significantly
was observed after 96 h of exposure to 0.1 μg/L DCOIT (Fig. 2A). A impaired by DCOIT exposure. After 24 h of exposure to the lowest
similar result was observed for digestive gland after 96 h of exposure to concentration of DCOIT tested (0.1 μg/L), CAT activity decreased in the
DCOIT (Fig. 2B). After 24 h, both concentrations of DCOIT exposure in gills of animals (Fig. 3C). In the digestive gland, the highest con-
the digestive gland reduced levels of SOD-like mRNA (Fig. 2B). centration of DCOIT tested was needed to decrease the activity of CAT
The mRNA levels of the microssomal GST (MGST-like) gene were after 96 h of exposure (Fig. 3D).

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H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

Fig. 3. Activity of superoxide dismutase (SOD) (A and B), catalase (CAT) (C and D), and glutathione S-transferase (GST) (E and F) in gills (left) and digestive glands
(right) P. perna exposed to 0.1 μg/L and 10 μg DCOIT. Different letters indicate statistically significant differences (p < 0.05).

The enzymatic activity of GST was significantly increased in di- post-DCOIT exposure. Neither gill nor digestive gland tissues showed
gestive gland tissues exposed to both concentrations of DCOIT com- altered levels of protein carbonylation after the exposure to either
pared to those exposed to control treatments after 24 h (Fig. 3F). No 0.1 μg/L or 10 μg/L DCOIT (Table 2).
significant differences (p > 0.05) were observed in gills tissues ex-
posed to DCOIT for either 24 h or 96 h (Fig. 3E).
4. Discussion
GSH is a non-enzymatic component of the antioxidant defence
system. A decrease in GSH levels was observed exclusively in the gills of
The current study provided new data regarding the effects of DCOIT
animals. After 96 h of exposure, GSH content was decreased in the gills
on the mussel species, P. perna. DCOIT is a widely used biocide that is a
when animals were exposed to 0.1 μg/L DCOIT (Fig. 4A). No differences
component of antifouling paints, and, to our knowledge, this is the first
were observed after 24 h of exposure (Fig. 4A).
study to show its effects on molecular and biochemical indicators of
adult mussel health. Although this booster biocide has been recognised
3.3. ROS and protein carbonyl content as a ‘Green Chemistry Challenge Award’ recipient for its environmental
safety properties (Chen and Lam, 2017), adverse effects have been
No significant changes in the levels of ROS were observed in gill or detected in organisms exposed to the compound (Do et al., 2018; Cima
digestive gland tissues after exposure to either concentration of DCOIT et al., 2008; Su et al., 2018; Su et al., 2019).
for either 24 or 96 h (Table 2). PCO levels also remained unchanged GSTs are important for providing a cellular defence response against

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H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

Fig. 4. Glutathione (GSH) content (mean ± standard error) in the gills (A) and digestive glands (B) of P. perna exposed to 0.1 μg/L and 10 μg/L DCOIT for 24 h and
96 h.

electrophilic agents in invertebrates (Freitas et al., 2019) and are cap- analyses performed are not specific for any particular isoform. More-
able of catalysing the conjugation of xenobiotics and endogenous sub- over, as reported by Trisciani et al. (2011), protein stability may lead to
strates to glutathione (GSH) (Leaver and George, 1998). Some studies differences between the mRNA levels and enzymatic activity. These
have demonstrated that GST activities increase in animals exposed to changes may also be due to the post-translational modification of pro-
DCOIT (Cima et al., 2013; Eom et al., 2019). In the present study, we teins (Regoli et al., 2011) or could be associated with the time frame
also observed an increase in GST activity in the digestive gland, how- analysed (Lüchmann et al., 2014).
ever, little is known about the relationship between observed increases Differences between levels of SOD activity and mRNA levels in the
in the levels of GST activity and the biotransformation of the com- two tissues analysed (gills and digestive gland) were also observed.No
pound. GSH is an important co-substrate that acts to detoxify electro- significant differences in the activities of enzimes were observed after
philic compounds such as xenobiotics and endogenous peroxides exposure to any concentration of DCOIT when compared to controls.
through reactions catalyzed by enzymes of the GST family (Townsend Despite the absence of lterations to SOD activity post-DCOIT exposure,
et al., 2003). In the present work, increases in GST activity were ob- SOD-like. mRNA levels were altered in both tissues (gills and digestive
served in mussels exposed to DCOIT and a decrease in GSH levels were glands), suggesting that the enzyme may be affected by DCOIT at the
also observed. DCOIT can also deplete glutathione content, mainly molecular level. Altered mRNA levels may be the result of cellular at-
because the biocide belongs to the isothiazolone group (Arning et al., tempts to restore antioxidant capacity in relation to superoxide levels.
2008; Collier et al., 1991). Studies have reported that the main me- ROS generation and protein carbonyl content were not affected by
chanism of DCOIT toxicity occurs through its passive diffusion across contaminant exposure. The absence of carbonyl protein induction by
biological membranes and interaction with GSH. DCOIT agrees with results showing that ROS levels were unaffected,
Previous data have indicated that increases in the GST activity occur since ROS are important inducers of the carbonylation process. Similar
when activities of other types of antioxidant defence systems decrease results regarding ROS levels were observed by Guerreiro et al. (2017)
(Manduzio et al., 2004). This is in accordance with our results, which and Barreto et al. (2018) who assessed the effects of another antifouling
showed altered levels of CAT activity in mussels exposed to DCOIT. This biocide capable of binding sulfhydryl groups (Chlorotalonil). These
enzyme is the major one responsible for the decomposition of hydro- authors observed that chlorothalonil was neither able to increase ROS
peroxides. However, it is important to note that GPx also has a similar levels in P. perna nor the Polychaete species, Laeonereis acuta.
activity, and can convert H2O2 to water through the oxidation of GSH to The exposure of organisms to antifouling biocides may produce
its corresponding disulfide, GSSG (Sorey, 1996). Although GPx activity oxidative stress. Several studies have shown that booster biocides, such
was not assessed in the present study, it is important to note that in- as chlorothalonil, dichlofluanid and irgarol (Barreto et al., 2018,
creased activity of the enzyme was observed in Perinereis aibuhitensis Guerreiro et al., 2020a, Lopes et al., 2020, Yamano and Morita, 1993,
exposed to DCOIT (Eom et al., 2019). Wang et al., 2013), for example, may alter the antioxidant defence
With the improvement of genomic technologies, molecular bio- system and/or levels of ROS in many aquatic organisms. For DCOIT,
markers can be used assess the effects of environmental contamination studies have also demonstrated its capacity to induce oxidative stress by
on aquatic organisms (Regoli et al., 2011). In this study, we evaluated significantly enhancing ROS production in Oryzias melastigma (Chen
SOD-like, MGST-like and GSTO- like mRNA levels. Even though GST et al., 2014b) and impairing maintenance of the energy supply of the
activity was altered in response to DCOIT exposure, only levels of the shrimp species, Litopennaeus vannamei (Su et al., 2019). It is important
MGST-like isoform were altered by DCOIT exposure. The MGST-like to note that some of the effects observed in the present study were
isoform is typically involved in glutathione (GSH) metabolism, pro- detected at the lowest concentration of DCOIT (0.1 μg/L), and that
tection against oxidative stress and eicosanoid metabolism (Johansson previous studies have used higher DCOIT concentrations.
et al., 2010) and GSTO is the isoform that is known to play a significant In the present study, we demonstrated that DCOIT is able to alter
role in the cellular response to oxidative stress (Won et al., 2011). the antioxidant defence system of mussels. Despite the fact that effects
mRNA levels of MGST-like gene were significantly decreased in the were observed in both the gills and digestive glands of animals, ob-
digestive gland after exposure to 10 μg/L, but not 0.1 μg/L, DCOIT after served changes were most prominent in the digestive gland. Trevisan
96 h. However, mRNA levels of the other isoform (GSTO-like) did not et al. (2016) and Guerreiro et al. (2020a) showed that gills may provide
differ in any tissues or timeframes analysed. Differences in data re- a first line of defence in mussels, since they have an increased capacity
garding GST expression and activity may occur because enzymatic to defend against environmental contaminants. Activities of defence

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H.B. Gabe, et al. Comparative Biochemistry and Physiology, Part C 239 (2021) 108870

Reactive Oxygen Species (ROS) and protein carbonyl (PCO) content in P. perna exposed to DCOIT. Data are expressed as mean ± standard error. Values are expressed for ROS as U Fluorescence x 107 and PCO as nmol per
enzymes are increased in the gills relative to the digestive glands of

2.02 × 109 ± 6.04 × 108

mussels. For instance, the activities of GST in M. edulis (Fitzpatrick

2.16 × 108 ± 7.0 × 107

et al., 1997) and SOD in P. perna (Almeida et al., 2005) are increased in

178,217 ± 27,040
gills relative to the digestive gland and levels of non-enzymatic anti-

72,667 ± 8346
oxidant defence peptide, GSH, of P. perna are also increased in the gills
versus the digestive gland (Guerreiro et al., 2020a). This is in ac-
cordance with results observed, because mussel gills are considered the
10 μg/L

main barrier between the organism and its environment (Ravera,

2001), and due to the enhanced antioxidant defence system of the gills,
only minor effects of toxic exposure were expected in the tissue.
2.68 × 109 ± 4.01 × 108
3.10 × 108 ± 8.0 × 107

5. Conclusion
128,468 ± 11,146

The present study provided new data regarding the effects of DCOIT
78,763 ± 8046

exposure on P. perna physiology. In general, we assessed the effects of

the biocide on enzymatic activity and gene expression in bivalves. Our
0.1 μg/L

results indicate that DCOIT modulates some components of the anti-

oxidant defence system of mussels, which could produce unfavourable
levels of oxidative stress, mainly because DCOIT can interact with
cellular thiols. This study has shown that the gills may act as a first line
of defence against exposure in mussels, since the tissue has higher levels
2.22 × 108 ± 5.71 × 107

2.97 × 109 ± 4.46 × 108

of antioxidant defence enzymes than the digestive gland.

161,326 ± 22,257

Declaration of competing interest

66,929 ± 12,014

The authors declare that they have no known competing financial

Control

interests or personal relationships that could have appeared to influ-

96 h

ence the work reported in this paper.

Acknowledgement
7.61 × 108 ± 3.49 × 107
5.8 × 107 ± 9.36 × 106

We would like to express special thanks to Dr. Igor Dias Medeiros for
providing gene sequences. This study was sponsored by the
100,395 ± 18,969

167,682 ± 24,438

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil

(CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento
Científico e Tecnológico – Brazil (CNPq; Proc. No 456372/2013-0) and
10 μg/L

FINEP – Pesquisa e Inovação (Proc. No 1111/13 – 01.14.0141.00). H. B.

Gabe and A. S. Guerreiro are graduate fellows of CAPES.
mg protein. No significant differences were observed by one-way ANOVA (p > 0.05).

References
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