An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse ‘Trojan Horse’ effects

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Combustion of biofuels from acidogenic fermentation in HCCI engines

<p>(A-D) Anti-tubulin immunohistochemical staining (Chromeo™ 488 conjugated secondary antibody) of digestive gland tissue sections from mussels exposed to different experimental conditions (A = Control; B = B[a]P 5 μg/L; C = B[a]P 50 μg/L; D = B[a]P 100 μg/L). (E) Quantitative fluorescence analysis of anti-tubulin immunoreaction. Data are mean ± SD of at least five replicates; * = p < 0.05 (Mann-Whitney <i>U</i>-test).</p

B[a]P-derived-DNA adduct levels, as measured by ³²P-postlabelling, in mussel digestive gland cells, after exposure to 0–1000 μg/L B[a]P for 3 days.

<p>Data are shown with the sexes combined (a) and separated (for those concentrations that resulted in detectable numbers of adducts)(b). All treatments had an n of 9 in total but the distribution of sexes was not even, therefore group n values are indicated in (b). Mismatching lowercase letters indicate differences between groups according to Tukeys HSD applied as post hoc tests following a generalised linear model (p< 0.05; n = 9). Insert: Representative autoradiographic profile of DNA adducts, measured by thin-layer chromatography ³²P-postlabelling, in digestive gland tissue exposed to B[a]P; no B[a]P-derived DNA adducts were detected in control (untreated) tissue (data not shown). The origin (OR), at the bottom left-hand corners, was cut off before exposure. The arrow indicates 10-(deoxyguanosin-<i>N</i>²-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-B[a]P (dG-<i>N</i>²-BPDE).</p

<i>Mytilus galloprovincialis</i> gene expression profiles of digestive gland tissue in animals exposed to B[a]P (5μg/L, 50μg/L and 100 μg/L).

<p>The heat map (A) (Pearson correlation, complete linkage algorithm) reports the log2 relative expression level with respect to the reference condition. 109 differentially expressed genes were generated in at least one condition. Microarray data were analyzed using the Linear Mode for Microarray Analysis (LIMMA) software. B statistics with adjusted p value, 0.05 and B.0 were used as threshold for rejection of the null hypothesis (no variation). Supporting information to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.g003" target="_blank">Fig 3</a> is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.s005" target="_blank">S2</a> Table and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.s007" target="_blank">S4 Table</a>. Venn diagram representation of gene expression patterns (Panel B) clearly depicted that no DEGs are shared between the three experimental conditions. All DEGs are obtained respect to the control condition. Data used to generate the Venn-diagram were obtained from microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.s007" target="_blank">S4 Table</a>). Four biological replications of were considered.</p

Effects of B[a]P on lipofuscin lysosomal content in the digestive gland cells of mussels, evaluated in cryostat tissue sections by the Schmorl reaction.

<p>Data, expressed as percent change with respect to control values, represent the mean ± SD of at least 10 replicates. * = p < 0.05 (Mann-Whitney <i>U</i>-test).</p

Over-representation analysis of DEGs in the digestive gland tissue of mussels exposed to B[a]P (5μg/L, 50μg/L and 100 μg/L).

<p>Showed are: experimental condition; biological processes; Number of up-regulated genes; Number of down-regulated genes. The over-represented biological processes in B[a]P-exposed animals <i>versus</i> control. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.s007" target="_blank">S4 Table</a>).</p

Chemical analysis of mussel tissues, after exposure to 0–100 μg L-1 BaP for 3 days.

<p>Chemical analysis of mussel tissues, after exposure to 0–100 μg L-1 BaP for 3 days.</p

Over-representation analysis of DEGs in the gills tissue of mussels exposed to B[a]P (5μg/L, 50μg/L and 100 μg/L).

<p>Showed are: experimental condition; biological processes; Number of up-regulated genes; Number of down-regulated genes. The over-represented biological processes in B[a]P-exposed animals <i>versus</i> control. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178460#pone.0178460.s006" target="_blank">S3 Table</a>).</p