Cellular pathways affected by carbon nanopowder-benzo(α)pyrene complex in human skin fibroblasts identified by proteomics.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

Carbon nanopowder acts as a Trojan-horse for benzo(α)pyrene in Danio rerio embryos.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the "particle-specific" effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

Structure and macromolecular composition of the zebrafish egg chorion.

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43 kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50 kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.

Identification and spatial distribution of the mRNA encoding an egg envelope component of the Cyprinid zebrafish, Danio rerio, homologous to the mammalian ZP3 (ZPC).

Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isolated a cDNA encoding a putative component of the zebrafish Danio rerio egg chorion, homologous to the mammalian ZP3 (ZPC). The predicted protein (zfZPC) has a calculated molecular mass of 58.4 kDa and contains a signal peptide (located in the N-terminal region) composed of 11 hydrophobic amino acid residues followed by a signal peptide cleavage site. The zfZPC contains the ZP domain, a characteristic amino acid sequence shared by all ZP proteins of the mammalian zona pellucida and of both amphibian and bird egg envelope components. The zfZPC also exhibits certain unique features including five N-terminal Q-rich tandem repeats presumably involved in the hardening of the chorion after the fertilization of the egg and a long C-terminal tail containing two potential sites of N-linked type glycosylation. RT-PCR and in situ hybridization revealed a restricted pattern of tissue distribution: the gene encoding zfZPC is transcribed only in the growing oocyte of sexually mature female fish.

Identification and spatial distribution of the mRNA encoding the gp49 component of the gilthead sea bream, Sparus aurata, egg envelope.

A cDNA encoding the precursor of one of the major components of gilthead sea bream, Sparus aurata, egg envelope has been cloned by reverse transcriptase polymerase chain reaction (RT-PCR) techniques. The clone was isolated starting from total RNA extracted from the liver of spawning female fish and estradiol-17 beta-treated male fish. Sequence analysis revealed that the cDNA encoded a protein of 405 aa corresponding to 49-kDa component (termed gp49), a glycoprotein belonging to the N-linked type. The gp49 protein is homologous to the Zl-3 of medaka Oryzias latipes, the mammalian ZPC and ZPC homologues of Xenopus laevis (xlZPC) and carp Cyprinus carpio (ccZPC). In addition, the open reading frame also encodes an additional aa sequence, the signal peptide, located in the N-terminal region of the protein. RT-PCR and in situ expression analyses evidenced an organ-restricted pattern: the mRNA was detected only in liver of spawning female and estradiol-17 beta-treated male fish but not in other tissues.

A new exon in the 5' untranslated region of the connexin32 gene.

The cloning and sequencing of two bovine connexin32 cDNAs are reported. Comparative analysis with known corresponding mammalian cDNA and protein sequences, besides confirming a high degree of similarity among these proteins, allowed us to identify some specific features of the bovine connexin32 gene. The latter include: the presence of a novel exon in the 5' UTR which is alternatively spliced, giving rise to a new mRNA species; the presence of two potential hairpin loops in the 5' and 3' UTR; and the presence of an additional amino acid, glycine235, in the C-terminal domain of the 284 residue protein. Among the common features, the presence of polypyrimidine clusters within the 3' UTR, containing a consensus sequence for a cis-acting element, is noteworthy. Expression of connexin32 mRNAs was analysed in 16 bovine tissues. Transcript analysis suggests the presence, in cattle, of an alternative downstream promoter.

Expression pattern of c-sis, c-fos and c-jun in human placenta and embryofetal organs.

The expression pattern of c-sis, c-fos and c-jun was investigated in placenta and embryofetal organ specimens from the first trimester. Northern analysis of the placentae showed c-sis transcripts and c-fos expression. Northern analysis of the same genes in embryofetal organs pointed to the brain as the only organ where consistent transcriptional activity could be observed. RT-PCR analysis of c-fos and c-jun in placentae, staged at four different time periods in pregnancy, allowed to detect the expected fragments in all cases. The same was true when c-fos and c-jun were analyzed at the 13th week of gestation in all the embryofetal organs.