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

ABSTRACT: BACKGROUND: Sewage sludge (SL) and hydrochar (HC) agricultural applications are nowadays a common practice to soil conditioning and crop fertilization, however safety concerns for human and environmental health due to the presence of toxic compounds, have been recently increased. OBJECTIVES: Our aim is to assess the safety for humans and the environment of SL and derived HC applications. METHODS: We applied proteomics and bioinformatics analyses to the cell cultures used for DR-CALUX® bioassay to identify differentially abundant proteins regulated upon exposure to SL and derived HC samples rather than only using the Bioanalytical Toxicity Equivalents (BEQs) obtained by the DR- CALUX® system itself. RESULTS: DR-CALUX® cells exposed to SL or HC showed a differential protein pattern of abundance dependent on the SL and HC extracts. The modified proteins were involved in antioxidant pathways, unfolded protein response and DNA damage that have close correlations with dioxin effects on the biological system and with cancer and neurological disorder onset. Moreover, other evidence suggests the enrichment of heavy metals in extracts according to cellular response. DISCUSSION: This work represents the next step of CALUX bioassay for screening polyhalogenated dibenzo-p-dioxins/furans and dioxin-like polychlorinated biphenyls in HC and SL and confirm to be successful in screening proteins which abundance is dependent on the SL and HC extracts in addition to the occurrence and biological activity of legacy toxic compounds not only belonging to organohalogens but still detectable for their biological activity in both matrices.

INSTRUMENT(S): autoflex

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture

SUBMITTER: Enxhi Shaba

LAB HEAD: Prof. Luca Bini

PROVIDER: PXD040115 | Pride | 2023-06-26

REPOSITORIES: Pride

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Project description:The primary goal of toxicology and safety testing is to identify agents that have the potential to cause adverse effects in humans. Unfortunately, many of these tests have not changed significantly in the past 30 years and most are inefficient, costly, and rely heavily on the use of animals. The rodent cancer bioassay is one of these safety tests and was originally established as a screen to identify potential carcinogens that would be further analyzed in human epidemiological studies. Today, the rodent cancer bioassay has evolved into the primary means to determine the carcinogenic potential of a chemical and generate quantitative information on dose-response behavior in chemical risk assessments. Due to the resource-intensive nature of these studies, each bioassay costs $2 to $4 million and takes over three years to complete. Over the past 30 years, only 1,468 chemicals have been tested in a rodent cancer bioassay. By comparison, approximately 9,000 chemicals are used by industry in quantities greater than 10,000 lbs and nearly 90,000 chemicals have been inventoried by the U.S. Environmental Protection Agency as part of the Toxic Substances Control Act. Given the disparity between the number of chemicals tested in a rodent cancer bioassay and the number of chemicals used by industry, a more efficient and economical system of identifying chemical carcinogens needs to be developed. Experiment Overall Design: Five-week old female B6C3F1 mice were exposed for 13 weeks in the following dose groups: 1) 1,5-Naphthalenediamine, CAS No. 2243-62-1, feed, 2000 ppm, positive lung carcinogen; 2) 2,3-Benzofuran, CAS No. 271-89-6, gavage, 240 mg/kg, positive lung carcinogen; 3) 2,2-Bis(bromomethyl)-1,3-propanediol, CAS No. 3296-90-0, feed, 1250 ppm, positive lung carcinogen; 4) N-Methylolacrylamide, CAS No. 924-42-5, gavage (water), 50 mg/kg, positive lung carcinogen; 5) 1,2-Dibromoethane, CAS No. 106-93-4, gavage (corn oil), 62 mg/kg, positive lung carcinogen; 6) Coumarin, CAS No. 91-64-5, gavage (corn oil), 200 mg/kg, positive lung carcinogen; 7) Benzene, CAS No. 71-43-2, gavage (corn oil), 100 mg/kg, positive lung carcinogen; 8) N-(1-naphthyl)ethylenediamine dihydrochloride, CAS No. 1465-25-4, feed, 2000 ppm, negative lung carcinogen; 9) Pentachloronitrobenzene, CAS No. 82-68-8, feed, 8187 ppm, negative lung carcinogen; 10) 4-Nitroanthranilic acid, CAS No. 619-17-0, feed, 10000 ppm, negative lung carcinogen; 11) 2-Chloromethylpyridine hydrochloride, CAS No. 6959-47-3, gavage (water), 250 mg/kg, negative lung carcinogen; 12) Diazinon, CAS No. 333-41-5, feed, 200 ppm, negative lung carcinogen; 13) Malathion, CAS No. 121-75-5, feed, 16000 ppm, negative lung carcinogen; 14) Corn oil control, gavage; 15) Water control, gavage; 16) Rodent diet control, feed. Feed animals were exposed 7 days/week and gavage animals were exposed 5 days/week (5 ml/kg). After 13 weeks, animals were euthanized and lungs were collected. The right lobe was used for microarray analysis. Microarray analysis was performed on the lungs of three to four mice per treatment group.

Project description:Atmospheric particulate matter (PM) is a recognized risk factor for the global burden of disease in human populations. We are presenting here the application of toxicogenomics in the evaluation of the toxic effects of organic content of atmospheric particle matter (PM), from urban and rural environments (city of Barcelona and village of La Pobla, NE Spain), using the developing zebrafish embryo. The main goal is to identify the metabolic pathways involved in the adverse effects observed in zebrafish embryos exposed to PM organic content from urban and rural environments, also allowing the selection of genes of interest that are differentially expressed. The relevance of particle size to the PM toxicity is also addressed. Indeed, the zebrafish embryos were exposed to PM of aerodynamic diameter larger than 7.2 μm and smaller than 0.5 μm (PM10 and PM0.5, respectively). PM0.5 concentrated biological and toxic activities linked to organic substances. Transcriptomic analyses showed strong induction of the AhR signalling pathway (a.k.a. dioxin-like activity) for embryos exposed to both rural and urban extracts, correlating with the concentrations of PAHs. Urban extracts, with strong contribution of traffic emissions, specifically de-regulated oxidative stress-related genes, as well as pancreatic and eye-lens specific genes, two organs known to be affected by air pollution in humans. Exposure to rural extracts, with high contribution of wood burning emissions, affected genes implicated in basic cellular functions, in agreement with their strong embryotoxicity. Extracts from rural and urban samples elicited both common and specific transcriptome responses, suggesting different potentially adverse outcomes depending on PM source and composition. The authors thank the financial support of the Spanish Ministry (project TEA-PARTICLE, grant number CGL2011-29621) and the Portuguese Foundation for Science and Technology for the doctoral grant of Sofia R. Mesquita (SFRH/BD/80710/2011) funded by the Program POPH-QREN through the Portuguese Ministry of Education and Science and the European Social Fund, and support through project PEst-C/MAR/LA0015/2013.

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

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