Preventing illegal seafood trade using machine-learning assisted microbiome analysis.

BACKGROUND: Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems). RESULTS: Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score. CONCLUSIONS: We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish.

Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12).

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Contaminants from dredged sediments alter the transcriptome of Manila clam and induce shifts in microbiota composition.

BACKGROUND: The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam's microbial communities. RESULTS: Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments' chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities. CONCLUSIONS: Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon.

An In Vivo Whole-Transcriptomic Approach to Assess Developmental and Reproductive Impairments Caused by Flumequine in Daphnia magna.

Among veterinary antibiotics, flumequine (FLU) is still widely used in aquaculture due to its efficacy and cost-effectiveness. Although it was synthesized more than 50 years ago, a complete toxicological framework of possible side effects on non-target species is still far from being achieved. The aim of this research was to investigate the FLU molecular mechanisms in Daphnia magna, a planktonic crustacean recognized as a model species for ecotoxicological studies. Two different FLU concentrations (2.0 mg L-1 and 0.2 mg L-1) were assayed in general accordance with OECD Guideline 211, with some proper adaptations. Exposure to FLU (2.0 mg L-1) caused alteration of phenotypic traits, with a significant reduction in survival rate, body growth, and reproduction. The lower concentration (0.2 mg L-1) did not affect phenotypic traits but modulated gene expression, an effect which was even more evident under the higher exposure level. Indeed, in daphnids exposed to 2.0 mg L-1 FLU, several genes related with growth, development, structural components, and antioxidant response were significantly modulated. To the best of our knowledge, this is the first work showing the impact of FLU on the transcriptome of D. magna.

Nitrate Regulates Maize Root Transcriptome through Nitric Oxide Dependent and Independent Mechanisms.

Maize root responds to nitrate by modulating its development through the coordinated action of many interacting players. Nitric oxide is produced in primary root early after the nitrate provision, thus inducing root elongation. In this study, RNA sequencing was applied to discover the main molecular signatures distinguishing the response of maize root to nitrate according to their dependency on, or independency of, nitric oxide, thus discriminating the signaling pathways regulated by nitrate through nitric oxide from those regulated by nitrate itself of by further downstream factors. A set of subsequent detailed functional annotation tools (Gene Ontology enrichment, MapMan, KEGG reconstruction pathway, transcription factors detection) were used to gain further information and the lateral root density was measured both in the presence of nitrate and in the presence of nitrate plus cPTIO, a specific NO scavenger, and compared to that observed for N-depleted roots. Our results led us to identify six clusters of transcripts according to their responsiveness to nitric oxide and to their regulation by nitrate provision. In general, shared and specific features for the six clusters were identified, allowing us to determine the overall root response to nitrate according to its dependency on nitric oxide.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Comparative toxicity assessment of alternative versus legacy PFAS: Implications for two primary trophic levels in freshwater ecosystems.

Perfluoroalkyl substances (PFAS) are common environmental pollutants, but their toxicity framework remains elusive. This research focused on ten PFAS, evaluating their impacts on two ecotoxicologically relevant model organisms from distinct trophic levels: the crustacean Daphnia magna and the unicellular green alga Raphidocelis subcapitata. The results showed a greater sensitivity of R. subcapitata compared to D. magna. However, a 10-day follow-up to the 48 h immobilisation test in D. magna showed delayed mortality, underlining the limitations of relying on EC50 s from standard acute toxicity tests. Among the compounds scrutinized, Perfluorodecanoic acid (PFDA) was the most toxic to R. subcapitata, succeeded by Perfluorooctane sulfonate (PFOS), Perfluorobutanoic acid (PFBA), and Perfluorononanoic acid (PFNA), with the latter being the only one to show an algicidal effect. In the same species, assessment of binary mixtures of the compounds that demonstrated high toxicity in the single evaluation revealed either additive or antagonistic interactions. Remarkably, with an EC50 of 31 mg L-1, the short-chain compound PFBA, tested individually, exhibited toxicity levels akin to the notorious long-chain PFOS, and its harm to freshwater ecosystems cannot be ruled out. Despite mounting toxicological evidence and escalating environmental concentrations, PFBA has received little scientific attention and regulatory stewardship. It is strongly advisable that regulators re-evaluate its use to mitigate potential risks to the environmental and human health.

Generation and characterization of cytochrome P450 3A74 CRISPR/Cas9 knockout bovine foetal hepatocyte cell line (BFH12).

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis.

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38ß MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38ß MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

The new PFAS C6O4 and its effects on marine invertebrates: First evidence of transcriptional and microbiota changes in the Manila clam Ruditapes philippinarum.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health.