Microplastics (MPs) and nanoplastics (NPs) have attracted considerable attention in the recent years as potential threats to the ecosystem and public health. This review summarizes current knowledge of pathological events triggered by micro- and nano-plastics (MP/NPs) with focus on oxidative damages at different levels of biological complexity (molecular, cellular, tissue, organ, individual and population). Based on published information, we matched the apical toxicity endpoints induced by MP/NPs with key event (KE) or adverse outcomes (AO) and categorized them according to the Adverse Outcome Pathway (AOP) online knowledgebase. We used existing AOPs and applied them to highlight formal mechanistic links between identified KEs and AOs in two possible scenarios: first from ecological, and second from public health perspective. Ecological perspective AOP based literature analysis revealed that MP/NPs share formation of reactive oxygen species as their molecular initiating event, leading to adverse outcomes such as growth inhibition and behavior alteration through oxidative stress cascades and inflammatory responses. Application of AOP on literature data related to public health perspective of MP/NPs showed that oxidative stress and its responding pathways, including inflammatory responses, could play the role of key events. However insufficient information prevented precise definitions of AOPs at this level. To overcome this knowledge gap, further mammalian model and epidemiological studies are necessary to support development and construction of detailed AOPs with public health focus.

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100. © 2016 SETAC.

Anesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L − 1 , EUG 30 mg L − 1 and MTMD 4 mg L − 1 . Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60-75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG were used. The degranulation of primary granules proved to be a useful assay for measuring the effects of stress on neutrophil function in fish. Eugenol and metomidate prevented stress-induced decrease of neutrophil function while MS 222 did not. RightsWorks produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted. AbstractAnesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L − 1 , EUG 30 mg L − 1 and MTMD 4 mg L − 1 . Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60-75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG w...

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A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,30,5,50-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content. Abstract A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3 0 ,5,5 0 -tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.

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Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease.