BioRisk | a 24 1-25 | (2022) . Cr ae a eae aes
doi: 10.3897/biorisk.17.77279 RESEARCH ARTICLE & B lO R IS k

https://biorisk.pensoft.net

Seasonal changes in the pro/antioxidant status of
mussels Mytilus galloprovincialis (Lamarck, 1819)
from Bulgarian Black Sea coastal habitats

Elina Tsvetanova', Almira Georgieva', Nesho Chipev*, Albena Alexandrova'

| Institute of Neurobiology, Bulgaria Academy of Sciences, 23, Acad. G. Bonchev str, 1113 Sofia, Bulgaria
2 Department of Biology, Shumen University, 115, Universitetska str., Shumen, Bulgaria

Corresponding author: Elina Tsvetanova (elinaroum@yahoo.com)

Academiceditor: Roumiana Metcheva | Received 29 October 2021 | Accepted 29 November 2021 | Published 21 April 2022

Citation: Tsvetanova E, Georgieva A, Chipev N, Alexandrova A (2022) Seasonal changes in the pro/antioxidant
status of mussels Mytilus galloprovincialis (Lamarck, 1819) from Bulgarian Black Sea coastal habitats. In: Chankova
S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17:
241-251. https://doi.org/10.3897/biorisk.17.77279

Abstract

The pro/antioxidant status of marine macrozoobentic organisms is being increasingly applied in environmental
monitoring and conservation programs. The oxidative stress level in marine bivalves can provide valuable infor-
mation not only on the health of the organisms and their populations, but also on the current state of habitats
and ecosystems. The aim of the present study was to make the first comprehensive investigation of the seasonal
changes in the antioxidant activity in different organs (gills, digestive gland and foot) of M. galloprovincialis from
representative Bulgarian Black Sea coastal habitats. The lipid peroxidation and glutathione levels, as well as ac-
tivities of the antioxidant enzymes catalase, superoxide dismutase, glutathion peroxidase, glutathion reductase,
glucose-6-phosphate dehydrogenase and glutathione-S-transferase of the organs were measured spectrophoto-
metrically. Our hypothesis was that enhanced environmental pressure during the summer season, induced by
multiple factors (biogenic, abiogenic and anthropogenic) led to weakening of the antioxidant protection in
mussels at the beginning of autumn. The reaction of the mussel organism to the multiple stress factors was
specific for the target organ and the type of the biomarker. Significant differences were present in the activity
of the antioxidant system in mussels from the northern and southern coastal locations. The seasonal changes in
the pro/antioxidant status of mussels were primarily due to specific seasonal changes in factors concerning the
marine environment at the concrete locality. Further research is obviously needed to confirm the present results
and provide a more complete data of seasonal and spatial changes in the antioxidant defense system of mussels

from the Bulgarian Black Sea coastal area and their implementation in biomonitoring programs.

Copyright Elina Tsvetanova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

242 Elina Tsvetanova et al. / BioRisk 17: 241-251 (2022)

Keywords

Antioxidant enzymes, Black Sea, glutathione, Mytilus galloprovincialis, seasonal changes

Introduction

Mussels M. galloprovincialis are key components of Bulgarian Black Sea ecosystems
(Petrova and Stoykov 2011). Being sedentary and sessile filter-feeders, they are used as
suitable bioindicators for chemical contamination and the state of the marine environ-
ment, as a whole (Kamel et al. 2014; Faggio et al. 2018; Giirkan 2020).

Oxidative stress (OS) is a universal expression of the reaction of organisms to en-
vironmental impacts (Steinberg 2012) and its induction in marine bivalves can be
used to assess the condition of the marine environment and the health of ecosystems.
Contamination of the marine environment with metals, polycyclic aromatic hydro-
carbons (PAH) and eutrophication can lead to an increase of cellular prooxidative
processes such as lipid peroxidation, protein oxidation, oxidative DNA damages and
to stimulation of the antioxidant protection of marine organisms (Gnatyshyna et al.
2014; Kamel et al. 2014). Thus, the assessment of the state of the marine environment
requires not only measuring concentrations of pollutants, but also the level of cellular
responses of marine bivalves to xenobiotics, overexploitation, and climatic changes
(Giirkan 2020). It is considered that OS biomarkers can provide an early warning for
deterioration of the environment by identification of cell biochemical changes before
effects at the organism, population or community level take place (Lushchak 2011).

The OS response of mussels can vary broadly due to seasonal variations of tempera-
ture, hypoxia, metabolic status of the animals themselves, gonadal ripening, food avail-
ability, hydrological cycle, as well as metal and PAH concentrations in seawater (Mirzaei
et al. 2016; Koudryashova et al. 2019). Depending on the local strength and duration
of impacts, the antioxidant system of mussels can be activated or inhibited with con-
sequences for the organism — adaptation or death, respectively. This, in turn, can affect
higher levels of ecological hierarchy, i.e. population, communities and ecosystems.

The aim of the present study was to make the first comprehensive investigation of the
seasonal changes in the pro/antioxidant status of different organs (gills, digestive gland
and foot) of M. galloprovincialis from representative Bulgarian Black Sea coastal habitats.

Materials and methods

Sampling

The mussels were hand-gathered from sublittoral rocks and other hard substrate at 1-6 m
depth in June and September (2017-2018) from 16 different sites along the Bulgarian
Black Sea coast (Fig. 1). Mussel samples were placed in clean thermostable containers
with seawater and transported to the laboratory where they were further processed.

Seasonal changes in the pro/antioxidant status of Mytilus 243

Code Location N E
NI = ~—N2 N2 Kaliakra 43.3719° 28.4587"
N3 St. Konstantin & Elena 43.2294° 28.0146°
N4 Kranevo 43.3229° 28.0669°
N5 Tulenovo 43.4909° 28.5853°
f N6 Varna harbor 43.1920° 27.9212°
{ N7 Galata 43.1709° 27.9436°
t $1 St. Ivan Island (farm) 42.4391° 27.6850°
f Black sea $2 St. Ivan Island (east) 42.4379° 27.6965°
$3 St. Ivan Island (west) 42.4367° 27.6874"
. $4 Chervenka cape 42.4380° 27.6557°
$5 Stomoplo bay 42.2897° 27.7649°
86 Sozopol town (port) 42.4244° 27.6934"
$7 Sozopol town (coastal rocks) 42.4251° 27.7003°
$8 Kolokita cape 42.4092° 27.7296°
$9 St. Parashkeva 42.3205° 27.7720°

Figure |. M. galloprovincialis sample locations along the Bulgarian Black Sea coast

Tissue preparation

Three mussel organs were studied separately. The mussels (n=8—10 for each site) were
immediately dissected and the gills, foot and digestive gland were removed. Each in-
dividual organ was frozen in liquid nitrogen and stored at —80 °C prior to the bio-
chemical assays. Afterwards, all organs were homogenized in 100 mM potassium phos-
phate buffer (pH 7.4) using Potter Elvehjem homogenizer fitted with a Teflon pestle
(Thomas Scientific, USA). To receive a post nuclear fraction for determination of lipid
peroxidation and glutathione levels, the homogenates were centrifuged for 10 min at
3000 g. A part of the fraction was re-centrifuged at 12 000 g for 20 min to obtain a
post mitochondrial supernatant used for measurement of the antioxidant enzyme ac-
tivities. All operations were performed at 4 °C.

Biochemical analysis

All tested antioxidant biomarkers were measured spectrophotometrically using com-
mercially available kits, purchased by Sigma-Aldrich Co. LLC (USA): Lipid Peroxida-
tion (MDA) Assay Kit MAK085, Glutathione Assay Kit CS0260, SOD Assay Kit-
WST 19160, Catalase Assay Kit CAT100, Glutathione Peroxidase Cellular Activity
Assay CGP1, Glutathione reductase Kit GRSA and Glutathione-S-Transferase Assay
Kit CS0410. The manufacturer’s working instructions were strictly followed. The pro-
tein concentration was measured according to Lowry et al. (1951) by using a standard
curve of bovine serum albumin as standard.

244 Elina Tsvetanova et al. / BioRisk 17: 241-251 (2022)

Statistical analyses

Statistical analyses of raw data were carried out by ANOVA with Bonferroni post hoc
test. Comparisons were made using Mann-Whitney test. Multidimensional Scaling
(MDS) was applied to detect meaningful underlying dimensions and to explain the
observed similarities and dissimilarities.

Results

The estimated values of the studied antioxidant indicators in the mussels’ organs are
presented in Tables 1-3. The tested biomarkers demonstrated significant variability
among the studied organs, localities and the two seasons. Lipid peroxidation (LPO)
did not show seasonal differences (Table 1-3). The only significant difference was the
higher LPO in September than in June in the organs of mussels from St. Ivan Island
(west). Mussels from this location were gathered in the vicinity of the quay and the
high LPO values measured were probably due to the intense floating of boats or some
kind of momentary pollution.

The seasonal patterns in the activities of the antioxidant enzymes are presented in
Table 1-3. No statistically significant seasonal differences were present in the activity
of superoxide dismutase (SOD) in the foot and gills (except the samples from Cher-
venka cape) of mussels from all studied locations. In the digestive gland, however,
significant seasonal differences were found in all samples from the northern locations
of the coastal area, while in the samples from the southern locations only in the diges-
tive gland of mussels from St. Ivan Island (west) and Sozopol coastal rocks, differences
were present. Significant seasonal differences in the catalase (CAT) activities in the
foot of mussels were found in the samples from the following northern coastal loca-
tions: Kavarna mussel farm, Kaliakra and Kranevo, as well as in the mussels from the
following southern coastal locations St. Ivan Island (west), Sozopol coastal rocks and
cape Kolokita. At all these five locations, CAT activity in the foot was decreased in
September compared to June. No seasonal differences in CAT activity were observed
in the gills in any of the mussels from all the studied locations, except from Tulenovo.
In the digestive gland, no seasonal differences were found between the mussels from
all northern locations. In the southern locations, higher CAT values in mussels were
measured in September compared to June.

The values of glutathione (GSH) and related enzymes demonstrated clear seasonal
variations. GSH showed significant seasonal differences in the foot of mussels from the
northern locations (Table 1). In samples from the southern locations, the only significant
seasonal difference in GSH was found in the foot of mussels from Sozopol port and Sto-
moplo bay, where significantly lower GSH in September was found compared to June.
Concerning gills, only at 3 locations (St. Konstantin and Elena, cape Chervenka and St.
Parashkeva) statistically significant increase of GSH in September was present (Table 2).
The most pronounced seasonal differences in GSH concentration were found in the di-

Seasonal changes in the pro/antioxidant status of Mytilus 245

Table 1. Seasonal changes in oxidative stress biomarkers in foot of Mytilus galloprovincialis from North-
ern (N) and Southern (S) coastal areas of the Bulgarian Black Sea (mean+SD; *- significant differences:
ps0.05*; ps0.01**; ps0.001***June vs September).

LPO GSH SOD CAT GPX GR G6PDH GST
(nM MDA/ (ng/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein)
mg prot)

Site June Sept June Sept June Sept June Sept June Sept June Sept June Sept June Sept
N1 1.27 0.95 1700 325 21.80 21.79 0.19 0.03 5.90 3.39 5.94 10.88 1640 19.65 18.66 65.58
+0.4 +0.2 +239** +69 +74 +448 +0.12* +0.01 +1.9 +01 +32 +432 +48 $5.9 +5.5* 424.6
N2 1.41 0.96 2298 548 19.12 25.61 0.27 0.06 9.38 9.92 3.70 1468 1833 22.01 15.79 159.91
+0.7,- 0.2 +294" 4108 +2.9 +£7.22 +0.06% +0.01 +46 +47 +09* +406 +5.7 +3.7 +14 +58.6
N3 1.02 1.19 2171 506 25.53 13.72 0.25 0.13 842 426 1.78 1745 31.61 7.18 16.56 74.62
+0.3 40.2 4457" +32 412.7 +28 +0.07 +0.05 +3.7 +08 +1.0** +47 +10.60** +1.04 +9.14* +35.6
N4 1.52 1.12 2352 578 21.82 25.59 0.21 0.07. 10.51 16.33 3.16 663 28.77 48.69 23.70 128.70
+0.6 +0.2 +921** +168 +5.1 +106 +0.06* +002 +42 419 +412 +02 +130 +£7.5 +7.2™ +640
N5 1.374 0.90 2364 794 24.23 2484 0.36 0.25 883 5.38 1.74 14.75 29.40 32.11 32.88 227.70
0.3 £0.2 +480** +379 +62 +78 +0.15 +0.06 41.96 41.1 +09°* +406 +9.1 +1.30 48.7" 4114.5
N6 140 1.89 1483 527 23.41 22.53 0.24 0.13 482 0.96 3.87 4.95 32.01 14.5 25.61 94.97
+0.27 +£0.38 +168** +138 +87 +2.7 +0.07 +0.02 +07 +035 +16 +130 +75 +3.0 +5.9* 439.6
N7 1.23 1.81 1407 480 1440 20.68 0.19 0.10 440 0.85 3.19 11.91 2404 22.17 23.40 113.71
+0.3 +0.3 +160** +169 +36 +64 +007 +004 +420 +01 +2.0* +711 +4.6 +£7.28 +6.5* +30.2
Sl 141 143 1100 1561 25.72 1130 040 0.26 3.87 7.81 3.45 4,3 15.03 18.35 19.50 105.06
+0.8 +0.6 311 106 +5.0 +02 +031 0.09 2.3 +2.2 +07 +03 +3.8 +0.9 +7.9°* +28.3
S2. 130 148 1410 740 28.51 20.89 0.71 0.22 266 7.97 3.63 3.35 13.63 14.55
F0.1 +0.5 737 +695 +142 +49 +046 +0.09 +0.3* +29 +06 +404 +047 +5.1
$3 0.82 3.18 2762 1720 19.19 12.55 0.71 0.21 3.26 7.11 3.94 1.00 13.94 12.9
+0.1* 40.8 +873 +983 445 +11 +£0.52 0.14 +0.6* 16 +13 +00 +1.1 +0.04
S4 0.85 2.68 2410 1583 19.37 7.53 0.67 0.37 3.76 9.77 19.73 6.70 19.73 21.27
+0.1 +15 +899 +45 +94 +07 +051 +002 +0.3* +07 +60* +18 +49 +08
$5 154 2.08 2419 1030 27.72 19.03 041 0.22 2.55 7.13 12.35 2.47 18.55 1689 23.99 73.68
+0.6 +£0.88 +10 +922 +87 +44 +053 +0.06 +0.9* 29) 2.3% 0.1 +5.0 +67 +464 +23.4
S6 184 212 2018 415 34.91 20.51 0.30 0.53 3.60 5.19 12.17 262 18.96 19.20 25.76 73.06
+05 +14 +824 +318 £165 £75 +£0.20 +016 +1.7 1.6 5.8* 2.0 5.5 +5.6 +£7.1** +19.6

S7 171 162 1501 586 33.9 16.89 1.03 0.23 2.71 446 16.87 3.23 16.87 17.47 74.57
+0.5 +06 +131 +508 +130 +46 +404°* +0.16 +0.3 1.9 4.8* 1.2 +3.9 +£10.2 +249
S8 1.14 2.27 1544 921 21.25 21.84 1.05 0.26 2.20 9.38 16.28 3.80 19.61 16.68 94.01
+0.4 +07 +69 +788 +1.1 +96 +0.5** +0.14 +0.2* +448 +£9.5* +0.9 +3.1 +4.8 +16.0

S9 142 242 1422 725 28.06 19.84 0.09 0.18 415 9.82 9.67 0.80 18.60 13.46 18.25 73.68
+0.5 +08 +209 +355 +9.9 +71 +0.03 +4004 +1.7% +46 +1.7* +02 +3.4 +26 +1.4°* +265

gestive gland. In almost all locations, the GSH concentration in the autumn samples was
significantly higher. The only exceptions were in mussels from Kavarna in the northern
coastal area, and Sozopol port and Sozopol coastal rocks in the southern area. The activity
of glutathione peroxidase (GPx) in the mussels’ foot from the northern locations did not
show seasonal variations, except for the mussels from Varna harbor and Galata cape, where
GPx activity was significantly decreased in autumn (Table 1). In the foot of mussels from
the southern locations, statistically higher activity of the enzyme was found in the autumn
samples, except for those from St. Ivan Island farm, Sozopol port and Sozopol costal rocks.
The GPx activity in the gills of all mussels from the northern locations, collected in Septem-
ber, was lower in comparison with those from June (Table 2). In mussels from the southern
locations the inverse dependence was observed — an overall higher activity of the enzyme in
September compared to June. A similar pattern was found for GPx activity in the digestive
gland, i.e. in the northern coastal samples the activity in autumn was lower than in sum-

246 Elina Tsvetanova et al. / BioRisk 17: 241-251 (2022)

Table 2. Seasonal changes in oxidative stress biomarkers in gills of Mytilus galloprovincialis from North-
ern (N) and Southern (S) coastal areas of the Bulgarian Black Sea (mean+SD; *-significant differences:
ps0.05*; ps0.01**; p<0.001***June vs September).

LPO GSH SOD CAT GPX GR G6PDH GST
(nM MDA/ (ng/mg pro- (U/mg pro- (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein)
mg prot) tein) tein)
Site June Sept June Sept June Sept June Sept June Sept June Sept June Sept June Sept
NI 5.45 5.71 730 629 20.5 25.7 0.53 0.25 10.28 6.78 7.65 469 44.54 16.03 39.03 64.03
+2.2 43.7) +210 +196 +61 +9.6 +£0.23 +0.12 +50 +245 +436 +27 +114 +60 411.7 +25.2
N2 3.85 5.05 607 761 19.1 23.3 0.38 0.24 13.76 1.76 7.75 10.93 46.05 13.24 37.62 70.49
+#2.1 +2.9 +4126 +206 +3.8 +8.0 +0.04 +0.30 +3.9* 40.18 +£2.79 +5.7) +41* +3.2 +67 +25.6
N3 5.09 5.25 758 1218 204 189 044 047 16.79 1.79 7.00 7.99 66.73 10.60 40.79 65.72
+2.7 +0.6 +154* +4237 +46 +1.7 +0.06 +0.04 +8.9** 40.55 +36 +3.9 +13.2** +1.2 £27.11 +13.5
N4 447 492 636 946 182 266 0.33 044 1469 454 9.90 641 51.54 640 42.82 99.25
+0.9 +1.2 +4162 +4220 +8.7 416.2 40.09 +0.29 +8.0* +£1.92 +04 +2.9 +13.9* 42.2 412.1 +45.1
N5 4.79 5.91 647 778 28.7 349 0.61 0.29 15.05 425 632 21.75 65.32 21.43 49.85 85.75
+130 £1.55 +150 +251 4£11.0 413.4 +0.24* +0.04 +7.5* +0.79 41.1% +43 +18.7°* +£7.11 413.9 +123
N6 6.42 7.10 547 583 20.2 349 0.55 0.50 13.31 0.80 16.01 15.78 59.85 20.64 63.29 76.59
+26 41.8 +117 +201 +46 +7.3 £0.12 +0.18 +4.3** +046 42.4 +3.3 £17.7** +58 +20.2 +20.1
N7 5.36 5.29 575 889 22.3 25.7 0.37 0.50 13.90 6.25 14.02 15.36 58.67 14.85 61.52 67.76
1.3 $1.3 485 +257 +104 49.8 +0.12 40.25 +56 +1.89 4140 +3.5 +18.6* 42.0 420.7 +24.6
Sl 433 2.18 730 1355 145 15.6 0.71 0.86 3.80 6.10 10.05 2.53 25.44 22.21 18.04 83.33
+1.7 9 41.1 +176 +4256 +62 41.1 +036 40.2 +24 +1.56 +2.4* +07 +84 +01 +3.8* +45.6
S2. 3.76 7.43 733 1230 216 183 1.09 0.69 2.08 5.04 12.17 4.92 22.17 20.66
+1.8 42.7. +30 +308 +06 +64 +0.77 40.22 +03 +£2.01 +1.1* +03 +0.9 +7.6
$3. 3.95 9.26 717 1085 25.8 5.3 0.58 095 240 611 9.93 1.25 19.93 18.38
+0.4* 40.2 +494 +236 41.3 +£1.79 +0.1 +£0.28 +02 41.24 +43.2* +0.3 +26 +1.6
S4 2.74 409 590 2436 36.7 11.8 0.85 0.74 173 806 7.88 2.04 1885 16.66
0.4 1.1 +91*** +4387 +6.2* +0.8 +£0.59 +0.11 +0.2* +0.14 +0.6* +05 41.61 +3.5
S5 442 7.13 551 1140 19.0 15.5 0.65 066 2.16 616 536 49 32.85 23.73 11.33 60.02
14 +23 +146 +419 +12.0 +47 +£0.27 +043 +0.8** +3.58 42.5 42.6 +12.7 +59 +3.8* +13.7
S6 7.61 689 407 798 245 20.2 069 0.78 3.24 443 113 46 39.24 27.82 14.64 118.33
+2.6 +3.2 +4134 +268 +20.9 +8.5 +0.13 +037 +09 +23 +£5.7 +3.2 +218 +9.8 +3.8°* +31.7

S7 2.89 5.3 704 1053 31.3 166 0.69 0.61 146 4.35 867 13.87 18.67 22.28 103.08
0.1 1.5 68 +488 +69 +7.0 +0.06 +0.14 +03 +265 42.7 +100 +2.2 +6.5 +423
S8 3.63 5.54 743 1277 285 13.8 0.93 0.45 1.53 5.52 8.07 2.71 18.07 33.50 63.57
1.6 1.9 +124 +835 +4.2* +455 +0.63 +0.27 +0.0* 42.11 +3.9 +1.6 #32. 41735 +34.6

S9 414 690 432 1483 11.0 15.1 045 0.52 3.22 416 13.76 9.67 39.08 22.96 14.46 64.49
132 1:9) 59" 706" 46.0P 15:5. O16 40127 7 41.3. £1.98. 32) 9 17.9 75 7.6, 3.0% 28.5

mer, and in the southern locations higher activity in June was present (Table 3). Significant
seasonal differences were also observed in the activity of glutathione reductase (GR). In the
foot of mussels sampled in September a significant increase compared to June was found
for all northern coastal locations (except Kavarna farm and Varna harbor) (Table 1). In con-
trast, in the foot of the sampled mussels in September from the southern locations, a sig-
nificant decrease in the enzyme activity was present, with the exception of the region of St.
Ivan Island (St. Ivan Island mussel farm, St. Ivan Island east and St. Ivan Island west), where
low GR values in both seasons were present. In gills, a statistically significant decrease in GR
in autumn samples was present for all southern locations (Table 2). For GR in the diges-
tive gland of the mussels a statistically significant decrease in the autumn was observed for
locations from the southern coastal area with the exception of Sozopol port. A statistically
significant higher activity of glutathione-S-transferase (GST) in the gills and digestive gland

was observed in autumn for mussels from all southern coastal locations. GST activity was

Seasonal changes in the pro/antioxidant status of Mytilus 247

Table 3. Seasonal changes in oxidative stress biomarkers in digestive gland of Mytilus galloprovincialis
from Northern (N) and Southern (S) coastal areas of the Bulgarian Black Sea (meantSD; *-significant
differences: ps0.05*; ps0.01**; ps0.001***June vs September).

LPO GSH SOD CAT GPX GR G6PDH GST
(nM MDA/ — (ng/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein)
mg prot)
Site June Sept June Sept June Sept June Sept June Sept June Sept June Sept June Sept
Nl 3.72. 3.76 335.25 325.33 636 15.20 1.80 0.80 10.22 1.50 122 11.86 22.25 1342 4409 26.91
+1.4 41.92 £213.19 469.78 +2.94* +2.84 +0.95 +0.56 +4.78*** 40.22 +692 +3.44 +6.10* +2.23 +5.17 +6.17
N2 2.34 2.95 207 548 6.29 20.13 0.77 0.77 13.17 182 7.04 7.58 18.16 1640 40.97 43.20
+0.9 +08 +28* +108 +1.9°* +454 +£0.27 +018 +2.7** 40.60 +441 +431 42.7 435 +99 +128
N3 2.52. 2.48 311 506 7.85 19.49 0.80 060 12.28 184 889 1867 25.95 477 50.08 29.38
+0.8 +04 +92* +320 «$5.9* +7.5 +0.18 +039 +2.1°* +054 4+3.2 +4442 +12** +06 +84 +58
N4 3.75 3.94 241 578 7.77 2464 105 1.16 13.39 3.67 1046 11.30 23.35 618 50.87 40.74
#25 +09 +54* F168 +2.6°* +24 +0.29 +043 +4.2°* +154 +46 +£5.87 +43* +05 412.8 424.0
N5 2.08 3.11 315 794 6.85 17.01 1.14 045 1434 466 891 11.53 23.19 433 53.42 38.98
+0.3 41.0 +45*** +379 +45.8* +7.7 +044 +026 +45*°* 40.78 +£1.8 +605 +78 41.0 +81 +119
N6 4.29 5.33 168 527 4.57 19.25 043 1.29 13.11 0.75 5.24 1652 19.73 623 70.76 37.99
+#2.30 41.7 +41* +138 43.3 46.1 +016 +044 +5.7°* +048 +1.4* +4412 +3.6* +£1.30 £7.7% +3.5
N7 3.03 3.55 156 480 5.11 1752 045 1.39 12.15 608 5.38 11.58 1648 6.23 64.20 32.64
+200 +0.7 +26* +169 +43*°* +46 +017 +080 41.7% 41.91 424 +233 +41* +20 +141 +9.5
SI 2.13 2.89 173 626 8.01 5.66 0.82 1.19 3.86 636 19.17 438 17.01 10.59 5.51 26,12
£0.9 1.2 55% A275 3h2.0. F2.857 FOD5© 02 12.7 +0.84 +6.2** +11 +46 +425 +42.7* +49
$2 212 2.92 171 530 8.81 3.07 0.59 0.84 142 463 15.09 5.02 15.09 13.57
+0.9 1.1 148 +289 +3.9 +1.2 +047 +£0.23 +0.1* +1.87 +404 +166 +03 +2.9
$32.38 7.62 135 511 749 3.06 0.14 1.04 119 625 1438 3.3 1438 6.88
F1.1* +0.2 46* £50 £2.6* +0.7 +0.04* +0.33 +0.2* +£1.25 +1.6* +4042 +13 +0.6
S4 3.51 3.94 203 700 8.3 4.55 0.18 0.87 1.26 675 1612 635 1612 17.91
+0.67 +15 475% +258 +26 +1.5 +0.07* 40.03 +0.2* +1.19 +1.0* +49 +08 +3.3
$5 3.12 3.69 186 584 412 3.65 0.25 0.77 148 3.36 15.99 3.99 17.43 21.88 10.19 45.08
406 41.2 +49" 4234 41.5 41.5 +£0.09 +057 +0.8* +£1.85 +3.4°* 42.09 +34 11.1 +44 418.9
S6 3.6 4.25 144430 286 768 483 1.06 1.21 188 3.26 1546 11.16 19.19 20.11 889 43.95

+11 +19 +100 =9+5.7. +1.0 +042 +034 +05 +155 +21 +4488 +47 +126 +1.8* 421.3
S7 2.18 3.31 188+48 369 12.7 3.79 0.31 1.06 1.68 2.66 17.78 7.82 1445 17.79 33.08
+1.0 +11 #2220 +41* +24 +401* +051 +01 +£1.34 +3.7% +408 +423 +125 #1211
S8 = 2.99 3.57 158 533 6.69 5.21 0.19 0.92 1.3+0.0 3.54 17.25 5.5 “1725, 15:39 45.85
#21 41.3 436% +256 +£1.15 +16 +0.07* +0.26 +1.78 +4.0°* 42.19 +3.3 +5.4 419.1

S9 3.07 3.62 197 633 3.64 401 033 118 168 3.75 16.08 640 15.64 16.34 4.98 38.34
+16 413 +53 4261 +14 +424 +0.09* +066 +05 41.73 +17" +217 429 +59 +16 +495

significantly higher in foot of the mussels gathered in September from all coastal locations
(Table 1) and in the gills and digestive gland of the mussels from the southern locations.

Significant changes were observed in the activity of glucose-6-phosphate dehydro-
genase (G6PDH) in all three examined organs, only in the mussels from the northern
coastal locations. All samples from September had lower enzyme activities in the gills
and digestive gland compared to those from June. In the autumn food samples, a sta-
tistically significant decrease was observed in the mussels from St. Konstantin & Elena
and Varna harbor (Table 1).

Multidimensional scaling was used to reveal the presence of meaningful underly-
ing dimensions in the data set and to explain the observed similarities and dissimi-
larities between the values of the measured OS markers and their seasonal and spatial
variations (Fig. 2). In the space plane of the first two dimensions several groupings of
OS biomarkers based on similarity and distance could be observed.

248 Elina Tsvetanova et al. / BioRisk 17: 241-251 (2022)

CATS! Sept

08 LPO/N/J une

LPO/S/Sept GSH/S/Sept
o O

0.4 CAT/N/J une

02 CAT/S/Sune

GSH/N/Sept eeeeys
2 une
ti UN Sept GSH/N/J une .

\
a
a

7 UOISUBLIG
o
ty

LPO/N/Sept
oa

12 -10 08 106 04 D2 OO O22 O4 O86 O8 10 12 14
Dimension 1
Figure 2. Two dimensional MDS plane of oxidative stress biomarkers in mussels — data for two seasons

from the locations of the northern (N) and southern (S) Bulgarian Black Sea coastal areas

Along the horizontal axis (Dimension 1) two broad groupings according to simi-
larity can be distinguished. On the left side of the dimensional plane, loose groups
of pro/antioxidant markers in mussels from the northern locations of the coastal area
were located and on the right side, those from the locations of the southern coastal
area were situated. Along the vertical axis (Dimension 2) at the lower part of the di-
mensional plane samples with higher levels of GSH and related enzymes of the excited
detoxification system were situated while on the upper side of the dimensional plane
mussel samples with higher levels of LPO and activated CAT were located, indicating
activated antioxidant system in mussels’ cells.

Discussion

Data on the seasonal variation in cell oxidative status of M. galloprovincialis of the Bul-
garian Black Sea part are scarce. Our results demonstrated for the first time the pres-
ence of extremely high variability in the intracellular oxidative processes and antioxi-
dant defense system functioning in foot, gills and digestive gland of mussels together
with well pronounced seasonal and spatial differences.

Seasonal changes in the pro/antioxidant status of Mytilus 249

The main result obtained was the presence of significant differences in the anti-
oxidative reaction of mussels from the northern and southern coastal regions. The ob-
served significant variation in the activity of the antioxidant defense system of mussels
strongly depended on the organs studied. This was due to the different composition
and function of the studied organs. The most tangible seasonal changes were found in
the digestive gland. Here, statistically significant seasonal differences were present in all
studied biomarkers. ‘The digestive gland constantly receives substances from the envi-
ronment, incl. various xenobiotics, which makes it particularly susceptible to impacts
(Livingstone 1981). The tissue specificity in the antioxidant response can be linked to
the different bioaccumulation capacity and associated concentrations and retention
times of xenobiotics (Benito et al. 2017).

Clear seasonal changes were observed in GSH concentration and in the activity of
GSH-related enzymes. GSH can neutralize a broad variety of reactive oxygen species
such as singlet oxygen, hydroxyl radical, superoxide, anion radical, hydrogen peroxide
(Gostyukhina and Andreenko 2015). The major advantage of GSH as an antioxidant is
its abundance in cells and the ability to be mobilized as soon as oxidative stress intensi-
fies (Gostyukhina and Andreenko 2015). In addition to the ability of GSH to directly
eliminate reactive oxygen species, it is also a co-substrate of GPx and GST. Specifically,
GST plays a role in binding xenobiotics (Mannervik and Danielson 1988). Our results
demonstrated a significant increase in GST activity in the autumn samples compared
to those in early summer. This was most probably due to increased pollution pres-
sure along the Bulgarian Black Sea coast from intensified touristic flow and municipal
wastewater effluents during the summer months which activated the enzyme (Farcy et
al. 2011). The changes in the activity of GPx were especially indicative — in September
samples its activity was significantly reduced compared to June in the mussels from all
the northern locations, while in the mussels from the southern locations its activity was
significantly increased. Similar observations were found for GR. Concerning GGPDH
in the gills and in the digestive gland, there was a significant decrease in all northern
samples from September compared to June, while in the mussels from the southern
coastal locations there were no significant differences. These differences were probably
due to the presence of multiple stressor effects, related for example to the pollutant
inflow by the Danube River (Dineva 2011; Doncheva et al. 2020). Results of the MDS
analysis confirmed the presence of real dissimilarities in the pro/antioxidant status in
mussels from the northern and southern locations. In addition, activation of the de-
toxification system of the mussels in response to local contamination of the marine
environment was also proved.

Conclusion

The reaction of the mussel organism to various seasonal abiotic, biotic and anthro-
pogenic stress factors was proved to be specific for the target organ and the type of
biomarker reactions. It can be summarized that mussels MZ. galloprovincialis were

250 Elina Tsvetanova et al. / BioRisk 17: 241-251 (2022)

constantly exposed and responded to fluctuations of local conditions of their natural
habitats of the Bulgarian Black Sea coast, i.e. specific seasonal changes in temperature,
hypoxia, hydrological cycle and the metabolic status of the mussels themselves. ‘This is
a fact that should be considered in the interpretation of results and data from biomoni-
toring studies. Further research is obviously needed in order to confirm the present
results and provide a complete picture of the observed relationships and dependences.

Acknowledgements

This work was supported by the Grant N° KP-06-N21/7 of the National Science
Fund, Bulgaria. We thank Black Sea Shells Ltd. for assistance in providing material

from mussels.

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