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This study investigated the spatio-temporal variability of microplastics (MPs) in the sediments of the River Thames (UK) catchment over 30 months (July 2019 - Dec 2021). The average MP concentration was 61 items kg -1 d.w., with fragments <1 mm being dominant and polyethylene (PE) the most common polymer. Adjacent land use influenced MP concentrations and types, with industrial sites showing particularly high levels and a prevalence of small beads and industrial polymers. MP concentrations generally decreased after higher winter flows, likely due to sediment rearrangement or winnowing. This study describes the seasonal concentrations and characteristics of MPs present in sediment from the River Thames catchment, and attempts to identify their likely origin. Further, the study provides new insights into the mobility and fate of MPs in riverine settings under varying flow conditions, which is vital given the predicted increases in flooding under various global heating scenarios., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Seagrass meadows are one of the world's most diverse ecosystems offering habitats for an extensive array of species, as well as serving as protectors of coral reefs and vital carbon sinks. Furthermore, they modify hydrodynamics by diminishing water flow velocities and enhancing sediment deposition, indicating the potential for microplastic accumulation in their sediments. The build-up of microplastics could potentially have ecological impacts threatening to ecosystems, however little is known about microplastic abundance and controlling factors in seagrass sediments. Here we investigated microplastic characteristics and abundances within sediments underlying four seagrass meadow sites on the Turneffe Atoll, Belize. Sediment cores were collected and sub-sampled to include a range of replicate surface sediments (0-4 cm) and depth cores (sediment depths 0-2, 2-5, 5-10, 10-20 and 20-30 cm). These were analysed using 25 μm resolution μFTIR, with spectral maps processed using siMPle software. Microplastics were prevalent across the sites with an abundance range (limit of detection (LOD) blank-corrected) of < LOD to 17137 microplastics kg -1 dw found on the east side of the atoll. However, their abundances varied greatly between the replicate samples. Polyethylene and polypropylene were the most commonly detected polymers overall, although the dominant polymer type varied between sites. There were no differences in the abundance of microplastics between sites, nor could abundance distributions be explained by seagrass cover. However, abundances of microplastics were highest in sediments with lower proportions of fine grained particles (clay, <4 μm) suggesting that hydrodynamics override seagrass effects. Additionally, no patterns were seen between microplastic abundance and depth of sediment. This suggests that microplastic abundance and distribution in seagrass meadows may vary significantly depending on the specific geographical locations within those meadows, and that more complex hydrodynamic factors influence spatial variability at a localised scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to assess the abundance of microplastics in the gastro-intestinal tracts of three commercially important fish species in the UK, to determine whether catch location, feeding habits and fish size influence the amount of microplastics within fish. Fish were collected from two rivers in the UK: the River Thames and the River Stour (East Anglia). Fish were collected from two sites in the River Thames and one site in the River Stour. Species selected were European flounder (Platichthys flesus), whiting (Merlangius merlangus), and Atlantic herring (Clupea harengus), and were chosen to represent benthic and pelagic feeding habits. Across all locations, 41.5 % of fish had ingested at least one microplastic particle (37.5 % of European flounder, 52.2 % of whiting, and 28.6 % of Atlantic herring). The average number by species was 1.98 (±3.50) microplastics/fish in European flounder, 2.46 (±3.10) microplastics/fish in whiting and 1.47 (±3.17) microplastics/fish in herring. There were no significant differences in the number or mass of microplastics in fish based on river, site, species or habitat. However, the number and mass of microplastics within benthic fish (European flounder) in the River Stour were significantly higher than in benthic fish from the River Thames. By number of microplastics, larger and heavier fish were more highly contaminated. This study enhances our understanding of microplastics in commercially important fish but highlights that fish contamination is not easily predicted by feeding habits or catch location alone. Exposure and uptake is likely to vary with changing environmental conditions. Fish size tends to be a good predictor of contamination, with larger fish generally containing more microplastics. This is the first study to directly compare concentrations of microplastics in fish from different UK rivers and the first evidence of microplastics in the River Stour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism., (© 2024. The Author(s).)

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano., (© 2023. Springer Nature Limited.)

Microplastics (MP) are emerging contaminants with the capacity to bind and transport hydrophobic organic compounds of environmental concern, such as polybrominated diphenyl ethers (PBDEs). The aim of this study was to investigate the ingestion of nylon (polyamide) MP alone and when associated with PBDEs and their effects on Chironomus sancticaroli larvae survival and microbiome structure. Survival, PBDE uptake and microbial community composition were measured in fourth instar larvae exposed for 96 h to BDEs- 47, 99, 100 and 153 in the presence and absence of 1% w/w MP in sediment. Microbiome community structures were determined through high throughput sequencing of 16S small subunit ribosomal RNA gene (16S rRNA). Initial experiments showed that larvae ingested MP faster at 0.5% w/w MP, while depuration was more efficient at 1% w/w MP, although retention of MP was seen even after 168 h depuration. No mortality was observed as a result of PBDEs and MP exposure. MP had a negative effect on PBDE concentration within larvae (η 2 = 0.94) and a negative effect on sediment concentrations (η 2 = 0.48). In all samples, microbial communities were dominated by Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Gammaproteobacteria. Bacterial alpha diversity was not significantly affected by PBDEs or MP exposure. However, the abundance of discrete bacterial taxa was more sensitive to MP (X 2 = 45.81, p = 0.02), and PBDE exposure. Our results highlight that C. sancticaroli showed no acute response to MPs and PBDEs, but that MPs influenced bacterial microbiome structure even after only short-term acute exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

In contrast to marine ecosystems, the toxicity impact of microplastics in freshwater environments is poorly understood. This contribution reviews the literature on the range of effects of microplastics across and between trophic levels within the freshwater environment, including biofilms, macrophytes, phytoplankton, invertebrates, fish and amphibians. While there is supporting evidence for toxicity in some species e.g. growth reduction for photoautotrophs, increased mortality for some invertebrates, genetic changes in amphibians, and cell internalization of microplastics and nanoplastics in fish; other studies show that it is uncertain whether microplastics can have detrimental long-term impacts on ecosystems. Some taxa have yet to be studied e.g. benthic diatoms, while only 12% of publications on microplastics in freshwater, demonstrate trophic transfer in foodwebs. The fact that just 2% of publications focus on microplastics colonized by biofilms is hugely concerning given the cascading detrimental effects this could have on freshwater ecosystem function. Multiple additional stressors including environmental change (temperature rises and invasive species) and contaminants of anthropogenic origin (antibiotics, metals, pesticides and endocrine disruptors) will likely exacerbate negative interactions between microplastics and freshwater organisms, with potentially significant damaging consequences to freshwater ecosystems and foodwebs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microplastic fibres (MPFs) and nanoplastics (NPs) have the potential to be hazardous to soil organisms. Understanding uptake into organisms is key in assessing these effects, but this is often limited by the analytical challenges to quantify smaller-sized plastics in complex matrices. This study used MPFs and NPs containing inorganic tracers (In, Pd) to quantify uptake in the earthworm Lumbricus terrestris. Following seven days exposure, tracer concentrations were measured in earthworms and faeces. Earthworms exposed to 500 μg MPFs/g soil retained an estimated 32 MPFs in their tissues, while at 5000 μg MPFs/g earthworms retained between 2 and 593 MPFs. High variation in body burdens of MPFs was linked to soil retention in earthworms and reduced faeces production, suggesting egestion was being affected by MPFs. NPs uptake and elimination was also assessed over a more extended time-period of 42 days. After 1 day, NPs were no longer detectable in faeces during the elimination phase. However, some retention of NPs in the earthworms was estimated, not linked to retained soil, indicating not all NPs were eliminated. MPFs and NPs uptake can be quantified in earthworms and both particle types can be retained beyond the depuration period, suggesting the potential for longer-term accumulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Plastic pollution is one of today's great environmental challenges. Research addressing the issue of plastic pollution is growing, improving our predictions of risk, and informing the development of long-term solutions and mitigations. Nonetheless, sufficient evidence already exists to show that immediate and widespread action must be taken to reduce plastic release to the environment, and thus limit future harm. Given the cross-sector and multi-stakeholder approach that will be required to address plastic pollution, it is essential that contrasting opinions and misconceptions are tackled with respect to the status of knowledge, relative importance of plastics as an environmental stressor, and measures to reduce or mitigate harm from plastics in the environment. This perspective article lays out some key considerations and recommendations for moving forward with respect to both research and action., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Nanomaterials (NMs) are thermodynamically unstable by nature, and exposure of soil organisms to NMs in the terrestrial environment cannot be assumed constant. Thus, steady-state conditions may not apply to NMs, and bioaccumulation modeling for uptake should follow a dynamic approach. The one-compartment model allows the uptake and elimination of a chemical to be determined, while also permitting changes in exposure and growth to be taken into account. The aim of the present study was to investigate the accumulation of Ag from different Ag NM types (20 nm Ag 0 NMs, 50 nm Ag 0 NMs, and 25 nm Ag 2 S NMs) in the crop plant wheat (Triticum aestivum). Seeds were emerged in contaminated soils (3 or 10 mg Ag/kg dry soil, nominal) and plants grown for up to 42 d postemergence. Plant roots and shoots were collected after 1, 7, 14, 21, and 42 d postemergence; and total Ag was measured. Soil porewater Ag concentrations were also measured at each sampling time. Using the plant growth rates in the different treatments and the changing porewater concentrations as parameters, the one-compartment model was used to estimate the uptake and elimination of Ag from the plant tissues. The best fit of the model to the data included growth rate and porewater concentration decline, while showing elimination of Ag to be close to zero. Uptake was highest for Ag 0 NMs, and size did not influence their uptake rates. Accumulation of Ag from Ag 2 S NMs was lower, as reflected by the lower porewater concentrations. Environ Toxicol Chem 2021;40:1861-1872. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Microplastics are an environmental issue of global concern. Although they have been found in a range of environments worldwide, their contamination in the terrestrial environment is poorly understood. The lack of standardised methods for their detection and quantification is a major obstacle for determining the risk they pose to soil environments. Here we present a systematic comparison of microplastic extraction methods from soils, taking into account the characteristics of the soil medium to determine the best methods for quantification. The efficiency of organic matter removal using hydrogen peroxide, potassium hydroxide and Fenton's reagent was measured. Soils with a range of particle size distribution and organic matter content were spiked with a variety of microplastic types. Density separation methods using sodium chloride, zinc chloride and canola oil were tested. Recovery efficiencies were calculated and the impact of the reagents on the microplastics was quantified using Attenuated Total Reflectance (ATR) Fourier Transform-Infrared (FTIR) spectroscopy. The optimal organic removal method was found to be hydrogen peroxide. The recovery efficiency of microplastics was variable across polymer types. Overall, canola oil was shown to be the optimal method for density separation, however, efficiency was dependent on the amount of organic matter in the soil. This outcome highlights the importance of including matrix-specific calibration in future studies considering a wide range of microplastic types, to avoid underestimation of microplastic contamination. We show here that methods for extracting microplastics from soils can be simple, cost-effective and widely applicable, which will enable the advancement of microplastic research in terrestrial environments.

In order to assess risks to the natural environment from microplastics, it is necessary to have reliable information on all potential inputs and discharges. This relies on stringent quality control measures to ensure accurate reporting. Here we focus on wastewater treatment works (WwTWs) and the complex sample matrices these provide. Composite samples of both influent and effluent were collected over a 24 h period on two separate occasions from eight different WwTWs across the UK. Sludge samples were taken on five occasions from five WwTWs. The WwTW treatments included activated sludge, trickling filter and biological aerated flooded filter with or without tertiary treatment. Using micro-FTIR analysis, microplastics ≥25 μm were identified and quantified. Procedural blanks were used to derive limits of detection (LOD) and limits of quantification (LOQ). Where values were above the LOQ, microplastics in the influent ranged from 955 to 17,214 microplastic particles/L and in the effluent from 2 to 54 microplastic particles/L, giving an average removal rate of 99.8%. Microplastics could be quantified in sludge at concentrations of 301-10,380 microplastics/g dry weight, this analytical method therefore revealing higher concentrations than reported in previous studies. The most common polymers present overall were polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET). We also report on critical considerations for blank corrections and quality control measures to ensure reliable microplastic analysis across different sample types., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ecosystems in remote regions tend to be highly specific, having historically evolved over long timescales in relatively constant environmental conditions, with little human influence. Such regions are amongst those most physically altering and biologically threatened by global climate change. In addition, they are increasingly receiving anthropogenic pollution. Microplastic pollution has now been found in these most remote places on earth, far from most human activities. Microplastics can induce complex and wide-ranging physical and chemical effects but little to date is known of their long-term biological impacts. In combination with climate-induced stress, microplastics may lead to enhanced multi-stress impacts, potentially affecting the health and resilience of species and ecosystems. While species in historically populated areas have had some opportunity to adapt to mounting human influence over centuries and millennia, the relatively rapid intensification of widespread anthropogenic activities in recent decades has provided species in previously 'untouched' regions little such opportunities. The characteristics of remote ecosystems and the species therein suggest that they could be more sensitive to the combined effects of microplastic pollution, global physical change and other stressors than elsewhere. Here we discuss how species and ecosystems within two remote yet contrasting regions, coastal Antarctica and the deep sea, might be especially vulnerable to harm from microplastic pollution in the context of a rapidly changing environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Microplastics were characterized in eight water treatment works (WTWs) in England and Wales (UK). Sources included river water, groundwater, and an upland reservoir. Water treatment varied from disinfection, filtration, sedimentation, and activated carbon techniques. At each WTW, five repeat samples of raw and potable water and two repeat sludge samples were taken over 5 months. Microplastics in water were captured on 10 μm filters and nonplastic materials digested in the laboratory. Microplastics ≥25 μm were analyzed using Fourier-transform infrared microscopy. Blanks revealed consistent polyethylene (PE), poly(ethylene terephthalate) (PET), and polypropylene (PP) contamination. Spike recoveries for 63-90 μm polyamide microplastics demonstrated 101% (standard deviation, SD 27%) and 113% (SD 15%) recovery for raw and potable waters and 52% (SD 13%) for sludge. Only four of the six WTWs sampled for raw water and only two of eight WTWs in their potable water had microplastics above the limit of quantification. Considering only the WTWs with quantifiable microplastics, then on average, 4.9 microplastic particles/L were present in raw water and only 0.00011 microplastic particles/L were present in potable water (99.99% removal). Values in waste sludge were highly variable. PE, PET, and PP were the most common polymers quantified in raw water and sludge, and polystyrene and acrylonitrile butadiene styrene were the most common polymers quantified in potable water.

Microplastics attract widespread attention, including for their potential to transport toxic chemicals in the form of plasticisers and associated hydrophobic organic chemicals, such as polybrominated diphenyl ethers (PBDEs). The aims of this study were to investigate how nylon (polyamide) microplastics may affect PBDE accumulation in snails, and the acute effects of nylon particles and PBDEs on survival, weight change and inherent microbiome diversity and community composition of the pond snail Lymnaea stagnalis. Snails were exposed for 96 h to BDEs-47, 99, 100 and 153 in the presence and absence of 1% w/w nylon microplastics in quartz sand sediment. No mortality was observed over the exposure period. Snails not exposed to microplastics lost significantly more weight compared to those exposed to microplastics. Increasing PBDE concentration in the sediment resulted in an increased PBDE body burden in the snails, however microplastics did not significantly influence total PBDE uptake. Based on individual congeners, uptake of BDE 47 by snails was significantly reduced in the presence of microplastics. The diversity and composition of the snail microbiome was not significantly altered by the presence of PBDEs nor by the microplastics, singly or combined. Significant effects on a few individual operational taxonomic units (OTUs) occurred when comparing the highest PBDE concentration with the control treatment, but in the absence of microplastics only. Overall within these acute experiments, only subtle effects on weight loss and slight microbiome alterations occurred. These results therefore highlight that L. stagnalis are resilient to acute exposures to microplastics and PBDEs, and that microplastics are unlikely to influence HOC accumulation or the microbiome of this species over short timescales., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Terrestrial environments are subject to extensive pollution by plastics and, based on the slow degradation of plastics, are likely to act as long term sinks for microplastic debris. Currently the hazards of microplastics in soil and the potential impacts on soil organisms is poorly understood. Particularly the role of particle characteristics, such a size or polymer type, in dose-response relationships for microplastics is not known. The aim of this study was to assess the ingestion and toxicity of nylon (polyamide) particles, in three different size ranges, to Enchytraeus crypticus in a soil exposure. Effects were also compared with those of polyvinyl chloride (PVC) particles, in a single size range. Nylon particle ingestion was confirmed using fluorescence microscopy, with greatest ingestion for particles in the smallest size range (13-18 μm). To investigate how particle size affected survival and reproduction, E. crypticus were exposed to nylon particles in two well-defined size ranges (13-18 and 90-150 μm) and concentrations of 20, 50, 90 and 120 g/kg (2-12% w/w). An intermediate nylon size range (63-90 μm) and a larger sized PVC particle (106-150 μm), both at 90 g/kg, were also tested. Survival was not affected by either of the polymer types or sizes. Reproduction was significantly reduced, in a dose-dependent manner, by the nylon particles at high exposure concentrations (>90 g/kg). Smaller size ranges (13-18 μm) had a greater effect compared to larger size ranges (>63 μm), with a calculated EC 50 for the 13-18 μm size range of 108 ± 8.5 g/kg. This greater hazard could be qualitatively linked with the ingestion of a greater number of smaller particles. This study highlights the potential for toxic effects of plastics in small size ranges to soil organisms at high exposure concentrations, providing understanding of the hazards microplastics may pose in the terrestrial environment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plastic pollution is distributed across the globe, but compared with marine environments, there is only rudimentary understanding of the distribution and effects of plastics in other ecosystems. Here, we review the transport and effects of plastics across terrestrial, freshwater and marine environments. We focus on hydrological catchments as well-defined landscape units that provide an integrating scale at which plastic pollution can be investigated and managed. Diverse processes are responsible for the observed ubiquity of plastic pollution, but sources, fluxes and sinks in river catchments are poorly quantified. Early indications are that rivers are hotspots of plastic pollution, supporting some of the highest recorded concentrations. River systems are also likely pivotal conduits for plastic transport among the terrestrial, floodplain, riparian, benthic and transitional ecosystems with which they connect. Although ecological effects of micro- and nanoplastics might arise through a variety of physical and chemical mechanisms, consensus and understanding of their nature, severity and scale are restricted. Furthermore, while individual-level effects are often graphically represented in public media, knowledge of the extent and severity of the impacts of plastic at population, community and ecosystem levels is limited. Given the potential social, ecological and economic consequences, we call for more comprehensive investigations of plastic pollution in ecosystems to guide effective management action and risk assessment. This is reliant on (a) expanding research to quantify sources, sinks, fluxes and fates of plastics in catchments and transitional waters both independently as a major transport routes to marine ecosystems, (b) improving environmentally relevant dose-response relationships for different organisms and effect pathways, (c) scaling up from studies on individual organisms to populations and ecosystems, where individual effects are shown to cause harm and; (d) improving biomonitoring through developing ecologically relevant metrics based on contemporary plastic research., (© 2019 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Daphnia magna were exposed to two pesticides in the presence or absence of microplastics (300 000 particles ml -1 1 µm polystyrene spheres) and to microplastics alone. The pesticides were dimethoate, an organophosphate insecticide with a low log Kow, and deltamethrin, a pyrethroid insecticide with a high log Kow. Daphnia were exposed to a nominal concentration range of 0.15, 0.31, 0.63, 1.25, 2.5, 5 mg l -1 dimethoate and 0.016, 0.08, 0.4, 2, 5 and 10 µg l -1 deltamethrin. Exposure to polystyrene microplastics alone showed no effects on Daphnia magna survival and mobility over a 72 h exposure. In the dimethoate exposures, mobility and survival were both affected from a concentration of 1.25 mg l -1 , with effects were seen on mobility from 28 h and survival from 48 h, with greater effects seen with increasing concentration and exposure time. In deltamethrin exposures, survival was affected from a concentration of 0.4 µg l -1 and mobility from a concentration of 0.08 µg l -1 . Effects of deltamethrin on mobility were seen from 5 h and on survival from 28 h, with greater effects on survival and mobility seen with increasing concentration and exposure time. Contrary to expectations, pesticide toxicity to Daphnia magna was not affected by the presence of microplastics, regardless of chemical binding affinity (log Kow). This therefore suggests that polystyrene microplastics are unlikely to act as a significant sink, nor as a vector for increased uptake of pesticides by aquatic organisms. CAPSULE: Polystyrene microplastics are unlikely to act as vector for increased uptake of pesticides by aquatic organisms., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microplastics are widespread throughout aquatic environments. However, there is currently insufficient understanding of the factors influencing ingestion of microplastics by organisms, especially higher predators such as fish. In this study we link ingestion of microplastics by the roach Rutilus rutilus, within the non-tidal part of the River Thames, to exposure and physiological factors. Microplastics were found within the gut contents of roach from six out of seven sampling sites. Of sampled fish, 33% contained at least one microplastic particle. The majority of particles were fibres (75%), with fragments and films also seen (22.7% and 2.3% respectively). Polymers identified were polyethylene, polypropylene and polyester, in addition to a synthetic dye. The maximum number of ingested microplastic particles for individual fish was strongly correlated to exposure (based on distance from the source of the river). Additionally, at a given exposure, the size of fish correlated with the actual quantity of microplastics in the gut. Larger (mainly female) fish were more likely to ingest the maximum possible number of particles than smaller (mainly male) fish. This study is the first to show microplastic ingestion within freshwater fish in the UK and provides valuable new evidence of the factors influencing ingestion that can be used to inform future studies on exposure and hazard of microplastics to fish., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Here we present the large-scale distribution of microplastic contamination in beach sediment across Europe. Sediment samples were collected from 23 locations across 13 countries by citizen scientists, and analysed using a standard operating procedure. We found significant variability in the concentrations of microplastics, ranging from 72±24 to 1512±187 microplastics per kg of dry sediment, with high variability within sampling locations. Three hotspots of microplastic accumulation (>700 microplastics per kg of dry sediment) were found. There was limited variability in the physico-chemical characteristics of the plastics across sampling locations. The majority of the microplastics were fibrous, <1mm in size, and blue/black in colour. In addition, using Raman spectrometry we identified particles as polyester, polyethylene, and polypropylene. Our research is the first large spatial-scale analysis of microplastics on European beaches giving insights into the nature and extent of the microplastic challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pollinators in agricultural landscapes can be exposed to mixtures of pesticides and environmental pollutants. Existing mixture toxicity modelling approaches, such as the models of concentration addition and independent action and the mechanistic DEBtox framework have been previously shown as valuable tools for understanding and ultimately predicting joint toxicity. Here we apply these mixture models to investigate the potential to interpret the effects of semi-chronic binary mixture exposure for three bee species: Apis mellifera, Bombus terrestris and Osmia bicornis within potentiation and mixture toxicity experiments. In the potentiation studies, the effect of the insecticide dimethoate with added propiconazole fungicide and neonicotinoid insecticide clothianidin with added tau-fluvalinate pyrethroid acaricide showed no difference in toxicity compared to the single chemical alone. Clothianidin toxicity showed a small scale, but temporally conserved increase in exposure conducted in the presence of propiconazole, particularly for B. terrestris and O. bicornis, the latter showing a near three-fold increase in clothianidin toxicity in the presence of propiconazole. In the mixture toxicity studies, the dominant response patterns were of additivity, however, binary mixtures of clothianidin and dimethoate in A. mellifera, B. terrestris and male O. bicornis there was evidence of a predominant antagonistic interaction. Given the ubiquitous nature of exposures to multiple chemicals, there is an urgent need to consider mixture effects in pollinator risk assessments. Our analyses suggest that current models, particularly those that utilise time-series data, such as DEBtox, can be used to identify additivity as the dominant response pattern and also those examples of interactions, even when small-scale, that may need to be taken into account during risk assessment.

Plastic debris is an environmentally persistent and complex contaminant of increasing concern. Understanding the sources, abundance and composition of microplastics present in the environment is a huge challenge due to the fact that hundreds of millions of tonnes of plastic material is manufactured for societal use annually, some of which is released to the environment. The majority of microplastics research to date has focussed on the marine environment. Although freshwater and terrestrial environments are recognised as origins and transport pathways of plastics to the oceans, there is still a comparative lack of knowledge about these environmental compartments. It is highly likely that microplastics will accumulate within continental environments, especially in areas of high anthropogenic influence such as agricultural or urban areas. This review critically evaluates the current literature on the presence, behaviour and fate of microplastics in freshwater and terrestrial environments and, where appropriate, also draws on relevant studies from other fields including nanotechnology, agriculture and waste management. Furthermore, we evaluate the relevant biological and chemical information from the substantial body of marine microplastic literature, determining the applicability and comparability of this data to freshwater and terrestrial systems. With the evidence presented, the authors have set out the current state of the knowledge, and identified the key gaps. These include the volume and composition of microplastics entering the environment, behaviour and fate of microplastics under a variety of environmental conditions and how characteristics of microplastics influence their toxicity. Given the technical challenges surrounding microplastics research, it is especially important that future studies develop standardised techniques to allow for comparability of data. The identification of these research needs will help inform the design of future studies, to determine both the extent and potential ecological impacts of microplastic pollution in freshwater and terrestrial environments., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Sewage effluent input and population were chosen as predictors of microplastic presence in sediments at four sites in the River Thames basin (UK). Large microplastic particles (1mm-4mm) were extracted using a stepwise approach to include visual extraction, flotation and identification using Raman spectroscopy. Microplastics were found at all four sites. One site had significantly higher numbers of microplastics than other sites, average 66 particles 100g -1 , 91% of which were fragments. This site was downstream of a storm drain outfall receiving urban runoff; many of the fragments at this site were determined to be derived of thermoplastic road-surface marking paints. At the remaining three sites, fibres were the dominant particle type. The most common polymers identified included polypropylene, polyester and polyarylsulphone. This study describes two major new findings: presence of microplastic particles in a UK freshwater system and identification of road marking paints as a source of microplastics., Capsule: This study is the first to quantify microplastics of any size in river sediments in the UK and links their presence to terrestrial sources including sewage and road marking paints., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Concern over reported honeybee (Apis mellifera spp.) losses has highlighted chemical exposure as a risk. Current laboratory oral toxicity tests in A. mellifera spp. use short-term, maximum 96 hour, exposures which may not necessarily account for chronic and cumulative toxicity. Here, we use extended 240 hour (10 day) exposures to examine seven agrochemicals and trace environmental pollutant toxicities for adult honeybees. Data were used to parameterise a dynamic energy budget model (DEBtox) to further examine potential survival effects up to 30 day and 90 day summer and winter worker lifespans. Honeybees were most sensitive to insecticides (clothianidin > dimethoate ≫ tau-fluvalinate), then trace metals/metalloids (cadmium, arsenic), followed by the fungicide propiconazole and herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). LC 50 s calculated from DEBtox parameters indicated a 27 fold change comparing exposure from 48 to 720 hours (summer worker lifespan) for cadmium, as the most time-dependent chemical as driven by slow toxicokinetics. Clothianidin and dimethoate exhibited more rapid toxicokinetics with 48 to 720 hour LC 50 s changes of <4 fold. As effects from long-term exposure may exceed those measured in short-term tests, future regulatory tests should extend to 96 hours as standard, with extension to 240 hour exposures further improving realism.

Background: Anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted., Results: In this study, we present the orthologs and phylogeny of 17 An. gambiae MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in An. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades., Conclusions: The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of An. gambiae and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species.

Background: Laboratory studies have demonstrated that a variety of immune signaling pathways regulate malaria parasite infection in Anopheles gambiae, the primary vector species in Africa., Methods: To begin to understand the importance of these associations under natural conditions, an association mapping approach was adopted to determine whether single nucleotide polymorphisms (SNPs) in selected immune signaling genes in A. gambiae collected in Mali were associated with the phenotype of Plasmodium falciparum infection., Results: Three SNPs were identified in field-collected mosquitoes that were associated with parasite infection in molecular form-dependent patterns: two were detected in the Toll5B gene and one was detected in the gene encoding insulin-like peptide 3 precursor. In addition, one infection-associated Toll5B SNP was in linkage disequilibrium with a SNP in sequence encoding a mitogen-activated protein kinase that has been associated with Toll signaling in mammalian cells. Both Toll5B SNPs showed divergence from Hardy-Weinberg equilibrium, suggesting that selection pressure(s) are acting on these loci., Conclusions: Seven of these eight infection-associated and linked SNPs alter codon frequency or introduce non-synonymous changes that would be predicted to alter protein structure and, hence, function, suggesting that these SNPs could alter immune signaling and responsiveness to parasite infection.

No studies have been performed on the mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that have a significant impact on malaria parasite transmission in endemic regions. In the present study, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells [ASE (Anopheles stephensi Mos. 43) cell line] from A. stephensi, a major vector of malaria in India, South-East Asia and parts of the Middle East. ASE cell mitochondria share many features in common with mammalian muscle mitochondria, despite the fact that these cells are of larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays as major a role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate-oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize proline at a rate comparable with that of alpha-glycerophosphate. However, the proline pathway appeared to differ from the currently accepted pathway, in that oxoglutarate could be catabolized completely by the tricarboxylic acid cycle or via transamination, depending on the ATP need.

It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.

The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization.

Polymorphonuclear leukocytes from healthy volunteers (HPMN) generated superoxide (O2*-) following treatment with various stimuli, such as phorbol myristate acetate (PMA), opsonized zymozan (OZ) and arachidonic acid (AA). Other types of n-3 polyunsaturated fatty acids (PUFAS), such as docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), also stimulated O2*- generation. The free form of DHA enhanced the generation of O2*- induced by PMA but inhibited that induced by OZ. In contrast, the ethylester of DHA (DHA-E) inhibited O2*- generation induced by PMA but stimulated that induced by OZ. Similar effects were also observed with ethylesters of EPA (EPA-E), DPA (DPA-E) and AA (AA-E). High concentrations of DHA-E reduced the PMA-induced formation of superoxide without affecting the cellular activity of protein kinase C (PKC). Similar phenomena were also observed with oral neutrophils from healthy volunteers (OPMN). These results indicate that PUFAS and their esters affect 02*- generation in human PMN via different pathways, thereby modulating inflammatory reactions.

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.

It has been shown previously that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, such as compactin, lovastatin, and pravastatin, block cholesterol synthesis, suppress lymphocyte functions, and beneficially affect atherogenesis. Recently, it was reported that compactin and lovastatin inhibit the respiratory burst of DMSO-differentiated HL-60 cells, an effect reversed by mevalonic acid. The mode of action of these inhibitors in this role is not understood fully. Thus, we studied the mechanism of inhibition of neutrophil superoxide (O2*-) generation by pravastatin and found that pravastatin at 0.5 mM inhibited the receptor-mediated tyrosine kinase (TK)-dependent pathway of O2*- generation and also luminol chemiluminescence but not the protein kinase C (PKC)-dependent or the TK- and PKC-independent pathways of O2*- generation in neutrophils. Pravastatin also inhibited the tumor necrosis factor-alpha- and formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of a tyrosine of a 115-kDa protein. These effects were not reversed by mevalonate. From these results it is concluded that pravastatin inhibited receptor-mediated O2*-generation by decreasing tyrosine phosphorylation but not by inhibiting the formation of an intermediate in the biosynthesis of cholesterol.

Human oral polymorphonuclear neutrophils (OPMN) generate reactive oxygen species even in the absence of stimulants. Because OPMN from newborn babies are exposed to colostrum and mature milk, the biological properties of these cells including the generation of reactive oxygen species might possibly be affected by the constituents of colostrum and milk. The present work reports the effects of colostrum and mature milk, including the effects of storage at low temperature, on superoxide generation by OPMN. Fresh colostrum and mature milk did not affect either endogenous or formyl-methionyl-leucyl-phenylalanine-induced generation of superoxide by OPMN. However, superoxide generation stimulated by phorbol myristate acetate or arachidonic acid was inhibited by colostrum and mature milk presumably due to binding of the ligands to milk proteins. During the storage of milk at 4 degrees C, free forms of unsaturated long-chain fatty acids increased, and there was concomitant increase in the ability of milk to generate superoxide radicals in OPMN. Kinetic analysis suggested that colostrum and mature milk regulate superoxide generation by OPMN, thereby modulating the bactericidal activity of these cells in the oral cavity., (Copyright 1999 S.Karger AG, Basel.)

Geranylgeranylacetone (GGA) induces apoptosis in human leukemia HL-60 cells in a dose- and time-dependent manner. This effect was completely prevented by the pan-caspase inhibitor z-Val-Ala-Asp(OMe) fluoromethylketone, thereby implicating the caspase cascade in the process. Prior to DNA fragmentation, GGA treatment markedly activated caspase-3(-like) proteases, which might be responsible for the observed apoptosis. In addition, GGA treatment interfered with the processing and membrane localization of Rap1 and Ras, and these changes may be a result of apoptosis. Moreover, nitric oxide donors significantly accentuated the GGA-induced apoptosis, suggesting that the apoptotic pathway induced by GGA might be regulated by a redox-sensitive mechanism. Taken together, these data suggest that the isoprenoid, GGA, is an effective inducer of apoptotic cell death in HL-60 cells.

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.

Nuclear breakdown leading to the formation of apoptotic bodies has been postulated to involve degradation of nuclear structural proteins, such as lamins A/C and B. Although nuclear segmentation occurs during the maturation of polymorphonuclear leukocytes (neutrophils), its mechanism is not known. We found that human neutrophils have lamin B but lack lamins A/C while mononuclear cells possess all three types of lamin as assessed by immunoblotting. Differentiation of human promyelocytic HL-60 cells into neutrophil-like cells was also accompanied by the down-regulation of lamins A/C but not of lamin B. Moreover, when compared with normal cells, neutrophils with the Pelger-Huët anomaly of nuclear hyposegmentation exhibited significantly lower activity of caspase-6, a lamin A/C-cleaving enzyme. Differentiated HL-60 cells showed higher activity of caspase-6 than that of untreated cells. These observations allow us to speculate that remodeling of nuclear lamins might underlie the mechanism for nuclear segmentation of neutrophils.

Lactoferrin (LF) is an iron-binding glycoprotein present in various secretions including milk and the specific granules of neutrophils. The main biological properties of this protein are thought to concern the regulation of iron absorption, antimicrobial activity and modulation of neutrophil activity. Copper bound LF (Cu-LF) inhibited the stimulation-dependent reduction of cytochrome c (Cyt. c) in guinea pig peritoneal neutrophils (GPMN) but were without effect on NADPH oxidase activity of the respiratory burst. However, Cu-LF stimulated the stimulation-dependent production of hydrogen peroxide as seen with superoxide dismutase (SOD). Similar but weaker inhibition of Cyt. c reduction than that shown by Cu-LF was observed with manganese-LF (Mn-LF) but not with ferrous-LF (Fe-LF) or apo-LF (Apo-LF). The inhibitory activity was concentration-dependent and the ID50s of Cu-LF and of Mn-LF were 0.1 and 5 microM, respectively. Reactive oxygen species (ROS) detected by luminol chemiluminescence (LCL) of stimulated-GPMN were partially inhibited by Cu-LF. Changes in LCL of stimulated GPMN induced by Cu-LF were similar to those of superoxide dismutase (SOD). Thus, it is concluded that low concentrations of Cu-LF had SOD-like activity and high concentrations of Cu-LF inhibited the stimulation-dependent generation of ROS.

Nitric oxide (NO) generated from 1-hydroxy-2-oxo-3, 3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-1 beta converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

A number of eicosanoids caused plasma membrane blebbing in hepatocytes and this could be inhibited in a dose-dependent fashion by the receptor antagonists AH6809 and ICI 192605. The pattern of effectiveness of eicosanoids interfering with canalicular vacuole accumulation in hepatocyte couplets differed from that causing blebbing; the two most effective eicosanoids here were PGD2 and PGF2 alpha.

Effects of nitric oxide (NO) and NO generating agents, on the electron transport system of mitochondria were examined in a study of the mechanism and physiological importance of NO in energy metabolism. In the presence of various substrates, uncoupled respiration was inhibited by NO in manner which was both dose- and oxygen tension-dependent. Simultaneously measuring changes in cytochrome absorption spectra and respiration showed that the site of action of NO is cytochrome oxidase. Similar inhibition was also brought about by 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO donor. Electron paramagnetic resonance (EPR) analysis revealed that inhibition of uncoupled respiration occurred only during the presence of NO in the reaction mixture. The inhibitory effect of NO was increased significantly by lowering the concentration of mitochondrial protein. No appreciable inhibition of respiration was observed in the presence of 3-morpholinosydnonimine (SIN-1), a peroxynitrite anion (ONOO-) generating reagent, but inhibition did occur in the presence of superoxide dismutase (SOD). These results indicate that NO reversibly interacts with mitochondria at complex IV thereby inhibiting respiration particularly under physiologically low oxygen tension and that de novo generated ONOO may have no significant effect under the present experimental conditions.

Effects of nitric oxide (NO) on oxygen uptake of Ehrlich ascites tumor cells (EATC) were examined in a study of the biological actions of NO on respiration and energy metabolism at the cellular level. Endogenous respiration of EATC was inhibited reversibly by NO in a dose dependent manner. Oxyhemoglobin, an NO trapping agent, restored the respiration promptly. The inhibitory action of NO also depended on oxygen-concentration, and the duration of suppression was prolonged remarkably at low oxygen tension. Similar inhibition was also observed in the presence of glucose. In this case, both lactate production and glucose consumption were promoted by NOC 18, an NO generating agent, and the activation was enhanced by lowering the oxygen-concentration. Furthermore, the membrane potential of EATC was depolarized transiently by adding NO, and the degree of depolarization was decreased in the presence of glucose. These results suggest that at physiologically low oxygen tension in ascites fluid, NO acts not only as a cytotoxic respiratory inhibitor but also as a regulatory factor in the energy metabolism of EATC.

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

We previously reported that phorbol 12-myristate 13-acetate (PMA)-induced superoxide (O2.-) generation of neutrophils was inhibited by hypericin, a photosensitizing pigment found in St. Johnswort (herb Hypericin triquetrifolium Turra), via a mechanism involving protein kinase C (PKC). To obtain further insights into the mechanism of inhibition, the effects of hypericin on stimulation-dependent O2.- generation and related enzymes of neutrophils were investigated. Hypericin inhibited O2.- generation of neutrophils induced by PKC-dependent and -independent stimuli in a light- and concentration-dependent manner. Oxygen was required for the light-dependent inhibition by hypericin. NADPH oxidase activity in a cell-free system and TNF-alpha-induced tyrosyl phosphorylation of neutrophil proteins were also inhibited by hypericin in a concentration- and light-dependent manner. However, tyrosine kinase of p60src, an enzyme not bound to a membrane, was not inhibited either in the light or in the dark. Oxygen uptake of neutrophils by photosensitization with hypericin resulted in the formation of singlet oxygen (1O2), O2.-, and hydroxyl radical (.OH) and enhanced lipid peroxidation. The formation of 1O2 was inhibited by azide, a quencher of 1O2, but not by desferrioxamine (DSF), a ferric ion chelator. By contrast, both generation of .OH and lipid peroxidation were inhibited by DSF but not by azide. Furthermore, PMA-induced O2.- generation inhibited by hypericin partially recovered in the presence of azide but not DSF. These results suggested that the light-dependent inhibition of O2.- generation by hypericin might be due to inhibition of tyrosine kinase, PKC, and NADPH oxidase via an oxygen-dependent mechanism, possibly through both Type I and II photosensitization mechanisms.

The photosensitizing effect of hypericin (HY), an antiretroviral agent, on the functions of isolated rat liver mitochondria has been investigated. The respiratory control ratio (RCR), ADP/O and membrane potential of mitochondria were decreased by HY in a light-dependent manner. Uncoupled respiration of mitochondria in the presence of succinate was also inhibited by HY in a light-dependent manner. The ID50 of hypericin for these inhibitions was approximately 0.5 microM. These inhibitory effects of HY were not observed when photosensitization was conducted under anaerobic conditions and were not affected by desferrioxamine (DSF) or superoxide dismutase (SOD). Upon photosensitization of HY, mitochondria consumed oxygen in the absence of respiratory substrate with concomitant formation of thiobarbituric acid reactive substance (TBARS). The amount of oxygen consumed was 100-times greater than that of TBARS formed. The oxygen uptake was partially inhibited by NaN3, and formation of TBARS was inhibited by DSF. Upon photosensitization of HY in the presence of mitochondrial membranes, the electron spin resonance (ESR) signal of 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO/.OH) was increased by a mechanism which was suppressed by DSF. An ESR signal for singlet oxygen bound to 2,5-dimethylfuran, 2,2,6,6-tetramethyl-4-piperidone (TEMP) was also detected under light in the presence of mitochondria. This signal of the TEMP-N-oxyl radical (TEMPO) was decreased by azide, which physically quenches singlet oxygen, but was increased by DSF. These results indicate that HY might inhibit mitochondrial functions by a type II photodynamic mechanism but that lipid peroxidation of biological membranes through an active oxygen-mediated photodynamic mechanism is not involved.

Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM). The alpha-tocopherol sensitive O2-. generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2-. generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2-. generation by neutrophils.

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O2-.) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

Hypericin, a photosensitizing plant pigment, has antiretroviral activity. When exposed to light, the inhibition of Friend leukemia virus (FLV)-induced splenomegaly by hypericin was increased. The ID50 was decreased to less than 2.5 micrograms/mouse by exposure to tungsten light (29 x 10(-3) W/cm2 for 3 min). This pigment inhibited phorbol 12-myristate 13-acetate (PMA)-activated protein kinase C of rat brain in a light- and concentration-dependent manner. The ID50 of hypericin and the light intensity for inhibition of PKC were 0.1 microM under a constant light of 29 x 10(-3) W/cm2 for 3 min and 5 x 10(-3) W/cm2 in the presence of 1 microM hypericin for 3 min, respectively. The PMA-induced respiratory burst of neutrophils was inhibited in the light but stimulated in the dark by hypericin. The ID50 for inhibition of the respiratory burst was similar to that for inhibition of PKC. These results suggest that hypericin might inhibited PKC-mediated processes of intact cells, including PMA-induced superoxide generation of neutrophils by some light-dependent mechanism, and that this mechanism might underlie its light-dependent inhibition of FLV infection.

Isolated hepatocytes incubated in the presence of either Ca2+ ionophore A23187 or thromboxane B2 develop many plasma membrane blebs which are a characteristic feature of toxic or ischaemic cell injury. When hepatocytes are incubated in the presence of both Ca2+ ionophore A23187 and any one of three thromboxane receptor antagonists (SK and F 88046, B.M. 13505, B.M. 13177), bleb formation is strongly inhibited. Hepatocytes incubated in the presence of both thromboxane B2 and any one of the three thromboxane receptor antagonists are also well protected from the formation of blebs. Treatment of isolated hepatocytes with Ca2+ ionophore A23187 is known to stimulate the production of thromboxanes. The data presented are consistent with thromboxane B2 acting as an intermediary in a proposed mechanism of cell injury and death in which elevated cytosolic free Ca2+ levels activate phospholipase A2 and the arachidonate cascade.