Proteomics coupled with AhR-reporter gene bioassay for human and environmental safety assessment of sewage sludge and hydrochar.

Today application of sewage sludge (SL) and hydrochar (HC) in agriculture is a common practice for soil conditioning and crop fertilization, however safety concerns for human and environmental health due to the presence of toxic compounds have recently been expressed. Our aim was to test the suitability of proteomics coupled with bioanalytical tools for unravelling mixture effects of these applications in human and environmental safety assessment. We conducted proteomic and bioinformatic analysis of cell cultures used in the DR-CALUX® bioassay to identify proteins differentially abundant after exposure to SL and the corresponding HC, rather than only using the Bioanalytical Toxicity Equivalents (BEQs) obtained by DR-CALUX®. DR-CALUX® cells exposed to SL or HC showed a differential pattern of protein abundance depending on the type of SL and HC extract. The modified proteins are involved in antioxidant pathways, unfolded protein response and DNA damage that have close correlations with the effects of dioxin on biological systems and with onset of cancer and neurological disorders. Other cell response evidence suggested enrichment of heavy metals in the extracts. The present combined approach represents an advance in the application of bioanalytical tools for safety assessment of complex mixtures such as SL and HC. It proved successful in screening proteins, the abundance of which is determined by SL and HC and by the biological activity of legacy toxic compounds, including organohalogens.

First Attempt to Couple Proteomics with the AhR Reporter Gene Bioassay in Soil Pollution Monitoring and Assessment.

A topsoil sample obtained from a highly industrialized area (Taranto, Italy) was tested on the DR-CALUX® cell line and the exposed cells processed with proteomic and bioinformatics analyses. The presence of polyhalogenated compounds in the topsoil extracts was confirmed by GC-MS/MS analysis. Proteomic analysis of the cells exposed to the topsoil extracts identified 43 differential proteins. Enrichment analysis highlighted biological processes, such as the cellular response to a chemical stimulus, stress, and inorganic substances; regulation of translation; regulation of apoptotic process; and the response to organonitrogen compounds in light of particular drugs and compounds, extrapolated by bioinformatics all linked to the identified protein modifications. Our results confirm and reflect the complex epidemiological situation occurring among Taranto inhabitants and underline the need to further investigate the presence and sources of inferred chemicals in soils. The combination of bioassays and proteomics reveals a more complex scenario of chemicals able to affect cellular pathways and leading to toxicities rather than those identified by only bioassays and related chemical analysis. This combined approach turns out to be a promising tool for soil risk assessment and deserves further investigation and developments for soil monitoring and risk assessment.

The cost of surviving nitrogen excess: energy and protein demand in the lichen Cladonia portentosa as revealed by proteomic analysis.

MAIN CONCLUSION: Different nitrogen forms affect different metabolic pathways in lichens. In particular, the most relevant changes in protein expression were observed in the fungal partner, with NO 3- mostly affecting the energetic metabolism and NH 4+ affecting transport and regulation of proteins and the energetic metabolism much more than NO 3- did. Excess deposition of reactive nitrogen is a well-known agent of stress for lichens, but which symbiont is most affected and how, remains a mystery. Using proteomics can expand our understanding of stress effects on lichens. We investigated the effects of different doses and forms of reactive nitrogen, with and without supplementary phosphorus and potassium, on the proteome of the lichen Cladonia portentosa growing in a 'real-world' simulation of nitrogen deposition. Protein expression changed with the nitrogen treatments but mostly in the fungal partner, with NO3- mainly affecting the energetic metabolism and NH4+ also affecting the protein synthesis machinery. The photobiont mainly responded overexpressing proteins involved in energy production. This suggests that in response to nitrogen stress, the photobiont mainly supports the defensive mechanisms initiated by the mycobiont with an increased energy production. Such surplus energy is then used by the cell to maintain functionality in the presence of NO3-, while a futile cycle of protein production can be hypothesized to be induced by NH4+ excess. External supply of potassium and phosphorus influenced differently the responses of particular enzymes, likely reflecting the many processes in which potassium exerts a regulatory function.