|Other Abstract||In recent years, the rapid development of coastal industry and the over-exploitation of marine resources have resulted in serious pollution in offshore and costal areas, inducing marine ecological risks. Different pollutants (such as organic pollutants and nanomaterials etc.) usually cause joint toxicity to marine organisms in a common or interactive manner. With excellent adsorption properties, graphene nanomaterials can interact with other pollutants after its release into the environment, thereby affecting the environmental behavior of these pollutants. As a typical organophosphate flame retardant (OPFR), triphenyl phosphate (TPP) has multiplex toxicities, including developmental toxicity and endocrine disruption. TPP can easily interact with graphene, as it has three benzene rings in the molecular structure. However, there is a lack of relevant research on the joint toxicities of graphene and TPP to marine organisms. The bivalve species, mussel Mytilus galloprovincialis has been often used as a bio-indicator in marine environmental assessment. Metabolomics, transcriptomics and traditional ecotoxicological methods were used to analyze the toxicity and its mechanisms of graphene and TPP in M. galloprovincialis. In addaition, the adverse output pathway (AOP) frameworks of immune and reproductive response of M. galloprovincialis were preliminarily constructed. Results were as follows:
1. Instrumental analysis of TPP in hepatopancreas
After exposed to graphene and TPP for 7 days, the concentrations of TPP in mussel hepatopancreas were 0.47 ± 0.16, 404.57 ± 258.22 and 770.77 ± 425.69 μg/g fat weight for the control group, TPP-exposed group and graphene + TPP co-exposed group, respectively. The concentrations of TPP in TPP-exposed group and graphene + TPP co-exposed group were significantly higher than that in the control group (P < 0.01). Compared with the TPP-exposed group, the co-exposed group had a higher average TPP concentration (about 1.9 times). These results implied that graphene might adsorb TPP and co-transferred into mussels, thereby resulting in increased accumulation of TPP in mussels.
2. Transcriptomic and metabolomic responses of M. galloprovincialis to graphene and TPP
After exposed to graphene and TPP for 7 days, hepatopancreas was used for transcriptomic and metabolomic analysis at molecular levels. Transcriptomic analysis revealed that the differentially expressed genes (DEGs) in hepatopancreas after graphene and TPP treatments were mainly involved in immune responses, endocrine disruption and reproductive development, substance and energy metabolism. The mechanisms of graphene-induced toxicity in hepatopancreas were analyzed based on the protein-protein interaction (PPI), indicating that graphene induced apoptosis or necrosis by up-regulating the expression of pro-apoptotic gene Caspase3, and activated cell transforming growth factor (TGF) and mitogen-activated protein kinase (MAPK) signaling pathway, and immune responses by activating Toll-like receptor signaling pathway. For energy metabolism, the DEGs involved in tricarboxylic acid (TCA) cycle (Idh3b) and sugar metabolism (Treh, Idh3b, Gpi, Ggt1 and Gusb, etc.) were up-regulated to enhance energy consumption in hepatopancreas after graphene exposure. Meanwhile, amino acids metabolism genes (Got2 and Ggt1) were up-regulated, enhancing osmotic regulation and maintaining intracellular glutathione (GSH) levels to alleviate the oxidative damage induced by graphene in M. galloprovincialis. In addition, graphene enhanced proliferation and differentiation of follicular cells by up-regulating the expression of Notch gene.
TPP induced immune defense and immune regulation in M. galloprovincialis by up-regulating the expression of immune and inflammatory genes (Xkr7, Fibcd1, Tp53i3 and Otud4). As for endocrine disruption and reproductive development, TPP affected the secretion by up-regulating the expression of Pck1 and Pck2, and inhibited the maturation of oocytes by down-regulating the expression of Cpeb1. In addition, the DEGs induced by TPP on material and energy metabolism of M. galloprovincialis hepatopancreas were similar to those induced by graphene, mainly affecting the sugar metabolism and amino acid metabolism genes.
Further analysis on the mechanisms of graphene + TPP-induced toxicity in hepatopancreas suggested that graphene + TPP activated Ras and NF-κB signaling pathways by up-regulating the expression of Notch and Birc genes, thereby inducing immune response in M. galloprovincialis. Meanwhile, graphene + TPP down-regulated the expression of Egfr, Birc3, Cdc42 and Smad3, thereby inhibiting the proliferation and differentiation of M. galloprovincialis hepatopancreas cells. As for endocrine disruption and reproductive development, graphene + TPP up-regulated the expression of EP300 to regulate the secretion of M. galloprovincialis, and affected the morphogenesis, proliferation and differentiation of germ cells through the Notch signaling pathway mediated by Dll1. In energy metabolism, the DEGs involved in tricarboxylic acid (TCA) cycle (Abat) and sugar metabolism genes (Gapdh, Sord and Eno1, etc.) were down-regulated to reduce energy consumption in M. galloprovincialis hepatopancreas after graphene + TPP exposure.
Meanwhile, metabolomic analysis found that graphene and TPP could affect several physiological processes in M. galloprovincialis hepatopancreas, such as energy metabolism and osmotic regulation. Metabolites and DEGs were used to analyze the perturbance of graphene in hepatopancreas. In the graphene-exposed group, the processes of glycolysis, glycine metabolism, succinate metabolism and tricarboxylic acid cycle were altered. Moreover, the contents of amino acids in M. galloprovincialis hepatopancreas were also involved in endocrine disruption. Metabolomic analysis revealed that metabolites correlated with energy metabolism (succinate), osmotic regulation (dimethylamine, glycine, homarine) and amino acid metabolism (threonine, glutamate, aspartate and alanine, etc.) were significantly changed after TPP treatment, indicating that TPP affected physiological processes in M. galloprovincialis, such as energy metabolism and osmotic regulation. In graphene + TPP-treated group, the alterations of glycolysis, urea cycle, serine metabolism, taurine metabolism and tricarboxylic acid cycle were observed in M. galloprovincialis hepatopancreas.
3. Immunotoxicity of graphene and TPP on M. galloprovincialis
After exposed to graphene and TPP for 7 days, traditional toxicological analysis methods were used to elucidate the immunotoxicity and the mechanisms for M. galloprovincialis induced by graphene, TPP and graphene + TPP at subcellular, cellular and gene levels. At subcellular level, transmission electron microscopy (TEM) observations showed that hemocytes could internalize graphene, thereby resulting in oxidative stress. At cellular level, oxidative stress could be induced by graphene and TPP in mussel hemocytes, which would further result in apoptosis, DNA damage and decrease in the lysosomal membrane stability (LMS). Increased oxidative stress and DNA damage in hemocytes were observed in the graphene-exposed group, but significantly reduced after graphene + TPP exposure. The up-regulated genes, including NF-κB, Bcl-2 and Ras, were mainly associated with reduced apoptosis and DNA damage after co-exposure to graphene and TPP. It seemed that the adsorption of TPP on graphene could inhibit the surface activity of graphene, thereby reducing its immunotoxicity. In addition, the physical damage in lipid bilayer and the increase of antioxidant enzyme activity were likely to be the early molecular event in the occurance of immune response, exhibiting individual’s immunosuppression and death as final adverse outcome. According to the systemic integration of the existing data resources, an AOP framework of immune response of M. galloprovincialis was established.
4. Reproductive and physiological responses of M. galloprovincialis to graphene and TPP
After exposed to graphene and TPP for 14 days, the physiological and reproductive effects and mechanisms of graphene and TPP to M. galloprovincialis were characterized. Oxidative stress was induced by graphene and TPP in hepatopancreas, which further caused damages in gill, hepatopancreas and gonad tissues. Compared with the control group, graphene significantly inhibited oxygen consumption rate (OR) of mussels and increased ammonia excretion rate (NR) (P < 0.05). The NR of M. galloprovincialis increased significantly after TPP exposure (P < 0.05). Compared with the graphene-exposed group, the oxidative stress, tissue damage and respiratory inhibition decreased markedly in graphene + TPP-treated group. Simultaneously, the expression levels in terms of reproductive genes (Vtg, Fkbp and Hsd) were markedly up-regulated after graphene + TPP exposure than those in the graphene-exposed group (P < 0.05). Also, the integrated biomarker response index (IBR) value in graphene + TPP treatment was lower than that in graphene and TPP treatments. In conclusion, the combined exposure of graphene and TPP exhibited lower physiological and reproductive toxicities on M. galloprovinciali, compared with the single exposure of graphene or TPP. In addition, the increase in the activity of antioxidant enzymes and the damage of the phospholipid bilayer were seemed to be the early molecular event in the occurrence of reproductive toxicity, exhibiting decline in individual reproductive capacity or population as the final adverse outcome. By integrating the existing data resources, an AOP framework about the occurrence of reproductive response of M. galloprovincialis was established.
Key words: Graphene, Triphenyl phosphate, Joint effects, Omics, Mytilus galloprovincialis|