Antagonistic interactions between benzo[a]pyrene and C60 in toxicological response of Marine Mussels

: This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C60) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC60, B[a]P and mixtures of nC60 and B[a]P into tissues was confirmed by GC-MS, LC-HRMS and ICP-MS. Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the toxic effect of B[a]P and C60. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C60, the majority of which were down-regulated (~52%). No DEPs were identified at any of the concentrations of nC60 (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC60), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis.

Preparation (FASP) method as described by [35]. The digested proteins were subsequently purified 172 using the STAGE tip procedure as previously described [36]. Tryptic peptides were analysed using 173 liquid chromatography-mass spectrometry (LC-MS).

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Peptide identification and quantification. Data analysis and quantification was performed using R 179 (Version 3.5.0) [38]. Thermo .raw files were imported into ProteoWizard [39] and converted to .mzML 180 format before identification using the MS-GF+ algorithm which is implemented in R via the 181 MSGFplus package [40]. MS-GF+ was chosen due to its known sensitivity in identifying more 182 peptides than most other database search tools and its ability to work well with diverse types of 183 spectra, configurations of instruments and experimetnal protcols [41]. The protein database utilised 184 in this study consisted of the UniProt KnowledgeBase (KB) sequences from all organisms from the 185 taxa Mollusca, sub category Bivalvia (84,410 sequences released 1/10/2018). This was cocatenated 186 with a common contaminants list downloaded from ftp://ftp.thegpm.org/f asta/cRAP (Version: 5 of 34 January 30 t h, 2015) using the R package seqRFLP [42]. Searches were carried out using the following 188 criteria: mass tolerance of 10 ppm, trypsin as the proteolytic enzyme, maximum number of clevage 189 sites = 2 and cysteine carbamidomethylation and oxidation as a fixed modification. Target decoy 190 approach (TDA) was applied as it is the dominant strategy for false discovery rate (FDR) estimation 191 in mass-spectrometry-based proteomics [43]. A 0.1 % peptide FDR threshold was applied in 192 accordance with standard practice, with a 1 % protein FDR applied after protein identification (via 193 aggregation). The resulting .mzid files were converted to MSnSet and quantified using label free were identified in more than two biological replicates. To quantitatively describe reliable and 206 biologically relevant protein expression changes based on single exposure to B[a]P, C60 or to a 207 combination of the two, the data analysis was split into three distinct sections. As per recent 208 recommendations, normalisation was carried out first [48]. Based on systematic evaluations of 209 normalisation methods in label free proteomics, normalisation between technical replicates was 210 carried out using variance stabilization normalisation (Vsn) [49]. Based on a study by Lazar et al. [48], 211 it was hypothesized the most likely cause of missing values will be due to a mixture of MAR (missing 212 at random), MCAR (missing completely at random) and MNAR (missing not at random) data. As 213 such, missing value imputation was carried out using a mixed methodology in the form of KNN (K 214 nearest neighbours, biological replicates) and QRILC (left censor method for MNAR data; whole 215 dataset) [50,51]. Following normalisation, differential expression was carried out using msmsTests

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[52] with p value less than 0.05 considered significant and Q-values (FDR: < 1%) calculated for p-217 value target matches with the Benjamini-Hochberg procedure. Enrichment of function among up-or 218 down-regulated proteins was calculated using GOfuncR using gene ontologies associated with 219 differentially expressed proteins (P-adj = 0.01, calculated using Benjamini-Hochberg method and q-220 value = 0.05). KEGG analysis was carried out on the identified unique proteins per treatment (p< 0.05) 221 using the clusterProfiler package [53]. KEGG annotation was performed using GhostKOALA [54] 222 and pathways with significant enrichment identified using ClusterProfiler (hypergeometric test, q < 223 0.05 following Benjamini correction

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The Pythagorean theorem method for combining standard errors was used to derive combined 288 standard errors for the predicted mean additive values (A) of C60 and BaP

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This enabled the 95% confidence limits to be derived for the predicted additive values. The   Table S1) of C60 301 dispersed in mussel-exposed seawater (~100 µg/mL) with brief ultrasonication followed by

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A rapid decline in the concentration of C60 in seawater was observed with time, with no 322 quantifiable amounts after day 1 (Table S3). At t0, the measured water concentrations are in 323 reasonable agreement with the nominal concentrations (427.6 ± 45.3, 63.8 ± 11.9 and 7.3 ± 1.8 µg L -1 324 for nominal concentrations of 1000, 100 and 10 µg L -1 respectively).

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analytical problem led to the loss of two samples of the gills from mussels exposed to Mix100 333 explaining the absence of standard error. at the lowest concentration. Regarding exposure to C60 only, higher DNA strand breaks compared to 339 the controls were observed only at the highest concentration (1 mg L -1 , p < 0.001). Lower C60 340 concentration did not appear to have any genotoxic effects on mussel digestive gland at the 341 concentrations tested. In mussels exposed to B[a]P + C60, significant higher DNA damage compared 342 to control were observed at all the tested concentrations. the digestive gland. Asterisks indicate the statistical differences observed between control and 346 exposed groups. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

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A significant increase (p = 0.00108) in 8-oxo-dGuo levels was detected in the digestive gland of 355 mussels exposed to C60 (15.3 ± 2.3) compared to control (         To provide further insight into the uptake of fullerenes by marine mussels, it was necessary to

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In a previous study in M. galloprovincialis [87], mussels exposed to C60 alone showed higher 555 accumulation of C60 in the digestive gland compared to the gill. Interestingly, co-exposure to 556 fluoranthene modified accumulation of C60, with higher accumulation of C60 when animals are 557 exposed to C60 alone compared to combined exposure.

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When comparing water and tissue concentrations for B[a]P and C60, the bioconcentration 559 observed in our conditions was much lower for C60 compared to B[a]P: the uptake in the DG of 560 mussels exposed to a similar aqueous concentration of B

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Further confirmation of the accumulation of fullerenes within mussels was afforded by ICP-MS 568 analysis of digestive gland tissues extracted from mussels exposed to Er-labelled fullerenes.

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However, no evidence for the presence of labelled fullerene aggregates within tissue sections using 570 our novel STEM-EDX approach was afforded, indicating that the fullerenes are likely distributed 571 within the tissues at the near molecular level (i.e. highly dispersed) and therefore below the 572 sensitivity of either microscopy or in situ spectroscopy approaches in complex materials such as these.

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In our study, the antagonistic effect observed in the co-exposure treatment at the highest 587 concentration may be caused by a reduction in ROS generation, or more effective scavenging of ROS 588 by C60, when C60 and BaP are present together in close association, as previously described by [9,72]. suggestion that mRNA directed protein synthesis is reduced in mussels exposed to higher B[a]P loads   769 that while mussels represent a target for environmental exposure to nanoparticles, exposure duration 770 may significantly contribute to NPs mediated toxicity [116]. As such, further work must be carried 771 out to explore mixture effects at different concentrations and over differing exposure duration.

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Supplementary Materials: The following are available online, Figure S1: Representative particle size 773 distribution showing the intensity-weighted hydrodynamic diameter (dH) of nC60 in mussel-exposed 774 seawater as determined by DLS (653±87 nm), Figure S2: (a) Bright-field TEM and (b) point EDX 775 spectroscopy analysis of Er3N@C80, Figure S3: Dark-field STEM and EDX spectroscopy mapping 776 analysis of Er3N@C80, confirming the necessity for spectroscopy to confirm the presence of labelled 777 fullerenes, using the characteristic X-rays emitted from Er upon electron irradiation, Figure S4: (a,c,e)

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Dark-field STEM and (b,d,f) corresponding point EDX spectroscopy analysis of cross-sections of 779 mussel digestive gland exposed to Er3N@C80, Table S1: The influence of benzo(a)pyrene (B[a]P) of the 780 hydrodynamic diameter (dH) of nC60 in mussel-exposed seawater as determined by DLS, Table S2:

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The concentration of B[a]P in seawater at T0, day 1 and day 3, Table S3: The concentration of nC60 in 782 seawater at T0, day 1 and day 3, Spreadsheet S1: Full list of DEPs, enriched Gene Ontology (GO) 783 terms and KEGGS pathways, R script S1: R script used for proteomics analysis.

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Factors influencing the partitioning and toxicity of nanotubes in the aquatic environment. Environ.