Distinct Molecular Processes Mediate Donor-derived Cell-free DNA Release From Kidney Transplants in Different Disease States.

BACKGROUND: Among all biopsies in the Trifecta-Kidney Study ( ClinicalTrials.gov NCT04239703), elevated plasma donor-derived cell-free DNA (dd-cfDNA) correlated most strongly with molecular antibody-mediated rejection (AMR) but was also elevated in other states: T cell-mediated rejection (TCMR), acute kidney injury (AKI), and some apparently normal biopsies. The present study aimed to define the molecular correlates of plasma dd-cfDNA within specific states. METHODS: Dd-cfDNA was measured by the Prospera test. Molecular rejection and injury states were defined using the Molecular Microscope system. We studied the correlation between dd-cfDNA and the expression of genes, transcript sets, and classifier scores within specific disease states, and compared AMR, TCMR, and AKI to biopsies classified as normal and no injury (NRNI). RESULTS: In all 604 biopsies, dd-cfDNA was elevated in AMR, TCMR, and AKI. Within AMR biopsies, dd-cfDNA correlated with AMR activity and stage. Within AKI, the correlations reflected acute parenchymal injury, including cell cycling. Within biopsies classified as MMDx Normal and archetypal No injury (NRNI), dd-cfDNA still correlated significantly with rejection- and injury-related genes. TCMR activity (eg, the TCMR Prob classifier) correlated with dd-cfDNA, but within TCMR biopsies, top gene correlations were complex and not the top TCMR-selective genes. CONCLUSIONS: In kidney transplants, elevated plasma dd-cfDNA is associated with 3 distinct molecular states in the donor tissue: AMR, recent parenchymal injury (including cell cycling), and TCMR, potentially complicated by parenchymal disruption. Moreover, subtle rejection- and injury-related changes in the donor tissue can contribute to dd-cfDNA elevations in transplants considered to have no rejection or injury.

Defining a natural killer cell-enriched molecular rejection-like state in lung transplant transbronchial biopsies.

In lung transplantation, antibody-mediated rejection (AMR) diagnosed using the International Society for Heart and Lung Transplantation criteria is uncommon compared with other organs, and previous studies failed to find molecular AMR (ABMR) in lung biopsies. However, understanding of ABMR has changed with the recognition that ABMR in kidney transplants is often donor-specific antibody (DSA)-negative and associated with natural killer (NK) cell transcripts. We therefore searched for a similar molecular ABMR-like state in transbronchial biopsies using gene expression microarray results from the INTERLUNG study (#NCT02812290). After optimizing rejection-selective transcript sets in a training set (N = 488), the resulting algorithms separated an NK cell-enriched molecular rejection-like state (NKRL) from T cell-mediated rejection (TCMR)/Mixed in a test set (N = 488). Applying this approach to all 896 transbronchial biopsies distinguished 3 groups: no rejection, TCMR/Mixed, and NKRL. Like TCMR/Mixed, NKRL had increased expression of all-rejection transcripts, but NKRL had increased expression of NK cell transcripts, whereas TCMR/Mixed had increased effector T cell and activated macrophage transcripts. NKRL was usually DSA-negative and not recognized as AMR clinically. TCMR/Mixed was associated with chronic lung allograft dysfunction, reduced one-second forced expiratory volume at the time of biopsy, and short-term graft failure, but NKRL was not. Thus, some lung transplants manifest a molecular state similar to DSA-negative ABMR in kidney and heart transplants, but its clinical significance must be established.

Environmental risk of nickel in aquatic Arctic ecosystems.

The Arctic faces many environmental challenges, including the continued exploitation of its mineral resources such as nickel (Ni). The responsible development of Ni mining in the Arctic requires establishing a risk assessment framework that accounts for the specificities of this unique region. We set out to conduct preliminary assessments of Ni exposure and effects in aquatic Arctic ecosystems. Our analysis of Ni source and transport processes in the Arctic suggests that fresh, estuarine, coastal, and marine waters are potential Ni-receiving environments, with both pelagic and benthic communities being at risk of exposure. Environmental concentrations of Ni show that sites with elevated Ni concentrations are located near Ni mining operations in freshwater environments, but there is a lack of data for coastal and estuarine environments near such operations. Nickel bioavailability in Arctic freshwaters seems to be mainly driven by dissolved organic carbon (DOC) concentrations with bioavailability being the highest in the High Arctic, where DOC levels are the lowest. However, this assessment is based on bioavailability models developed from non-Arctic species. At present, the lack of chronic Ni toxicity data on Arctic species constitutes the greatest hurdle toward the development of Ni quality standards in this region. Although there are some indications that polar organisms may not be more sensitive to contaminants than non-Arctic species, biological adaptations necessary for life in polar environments may have led to differences in species sensitivities, and this must be addressed in risk assessment frameworks. Finally, Ni polar risk assessment is further complicated by climate change, which affects the Arctic at a faster rate than the rest of the world. Herein we discuss the source, fate, and toxicity of Ni in Arctic aquatic environments, and discuss how climate change effects (e.g., permafrost thawing, increased precipitation, and warming) will influence risk assessments of Ni in the Arctic.

Mechanism of copper nanoparticle toxicity in rainbow trout olfactory mucosa.

Chemosensory perception is crucial for fish reproduction and survival. Direct contact of olfactory neuroepithelium to the surrounding environment makes it vulnerable to contaminants in aquatic ecosystems. Copper nanoparticles (CuNPs), which are increasingly used in commercial and domestic applications due their exceptional properties, can impair fish olfactory function. However, the molecular events underlying olfactory toxicity of CuNPs are largely unexplored. Our results suggested that CuNPs were bioavailable to olfactory mucosal cells. Using RNA-seq, we compared the effect of CuNPs and copper ions (Cu2+) on gene transcript profiles of rainbow trout (Oncorhynchus mykiss) olfactory mucosa. The narrow overlap in differential gene expression between the CuNP- and Cu2+-exposed fish revealed that these two contaminants exert their effects through distinct mechanisms. We propose a transcript-based conceptual model that shows that olfactory signal transduction, calcium homeostasis, and synaptic vesicular signaling were affected by CuNPs in the olfactory sensory neurons (OSNs). Neuroregenerative pathways were also impaired by CuNPs. In contrast, Cu2+ did not induce toxicity pathways and rather upregulated regeneration pathways. Both Cu treatments reduced immune system pathway transcripts. However, suppression of transcripts that were associated with inflammatory signaling was only observed with CuNPs. Neither oxidative stress nor apoptosis were triggered by Cu2+ or CuNPs in mucosal cells. Dysregulation of transcripts that regulate function, maintenance, and reestablishment of damaged olfactory mucosa represents critical mechanisms of toxicity of CuNPs. The loss of olfaction by CuNPs may impact survival of rainbow trout and impose an ecological risk to fish populations in contaminated environments.

Vape flavourants dull sensory perception and cause hyperactivity in developing zebrafish embryos.

E-cigarette use (vaping) during pregnancy has been increasing, and the potential exists for the developing brain in utero to be exposed to chemical constituents in the vape. Vapes come in over 7000 unique flavours with and without nicotine, and while nicotine is a known neurotoxicant, the effects of vape flavouring alone, in the absence of nicotine, on brain function are not well understood. Here, we performed a screen of vape aerosol extracts (VAEs) to determine the potential for prenatal neurotoxicity using the zebrafish embryo photomotor response (PMR)-a translational biosensor of neurobehavioural effects. We screened three commonly used aerosolized vape liquids (flavoured and flavourless) either with or without nicotine. No neurobehavioural effects were detected in flavourless, nicotine-free VAEs, while the addition of nicotine to this VAE dulled sensory perception. Flavoured nicotine-free VAEs also dulled sensory perception and caused hyperactivity in zebrafish embryos. The combination of flavour and nicotine produced largely additive effects. Flavoured VAEs without nicotine had similar neuroactive potency to nicotine. Together, using zebrafish PMR as a high throughput translational behavioural model for prenatal exposure, our results demonstrate that e-cigarette flavourants that we screened elicit neurobehavioural effects worthy of further investigation for long-term neurotoxic potential and also have the potential to modulate nicotine impact on the developing brain.

Municipal wastewater effluent exposure disrupts early development, larval behavior, and stress response in zebrafish.

While wastewater treatment standards have been progressively increasing, emerging contaminants such as pharmaceuticals can nonetheless pass through treatment and end up in our watersheds. Pharmaceuticals in the parts-per-billion range can impact fish behavior, survival, and recruitment in the wild. However, the ecological risk posed by whole municipal wastewater effluents (MWWE), a complex mixture, is not clear. This knowledge gap is particularly evident for early lifestages (ELS) of fish, and because effluent discharge events are typically short, the effects of short-term MWWE exposures to ELS fish are particularly important from an environmental perspective. Here we tested the effects of rapid 30-min exposures, and short-term 24- and 72-h exposures to MWWE on development, behaviors, and stress response in zebrafish (Danio rerio) embryos, larvae, and juveniles. We obtained 24-h composite samples of tertiary-treated MWWE that contained a mixture of chemicals with affinities for serotonergic, adrenergic, dopaminergic, and ion-channel receptors. Embryos exposed to 5%, 10%, and 50% MWWE experienced developmental delays in somitogenesis and hatching rate, although there was no effect on survival. Embryonic photomotor responses were affected following 30-min and 24-h exposures to 10% and 50% MWWE, and larval visual motor responses were reduced from 24-h exposure to 10% MWWE. Exposure to 10% MWWE dulled the juvenile cortisol and lactate response following an acute air-exposure. Compromised behavioral and stress performances demonstrate the capacity of MWWE to impact phenotypes critical to the survival of fish in the environment. Taken together, we found that zebrafish were sensitive to toxic effects of MWWE at multiple life-stages.

A rapid zebrafish embryo behavioral biosensor that is capable of detecting environmental ß-blockers.

ß-Blockers (BB) are one of the most commonly prescribed pharmaceuticals used for treating cardiovascular and acute anxiety-related disorders. This class of drugs inhibit ß-adrenoceptor signalling and given their growing, widespread use, BB are routinely detected in surface waters at nM concentrations. This is concerning as trace levels of BB impart developmental and reproductive dysfunction in non-target aquatic organisms, with potential for ecological risks. To date, environmental pharmaceutical risks to non-target animals are not part of the monitoring framework due to the lack of bioassays for assessing their biological effects. Behavioral endpoints have the advantage of a systems-level integration of multiple sensory signals and motor responses for toxicity screening; however, they are not currently used for risk assessment of environmental contaminants. The zebrafish (Danio rerio) embryo photomotor response (zfPMR) has been used in high-throughput behavioral screenings for neuroactive drug effects at high, therapeutic concentrations. Our objective here was to examine if we could utilize the zfPMR for screening environmental levels of BB. Embryos were placed into 96-well plates, exposed to chemicals and/or municipal wastewater effluent (MWWE), and their zfPMRs were measured with video-analysis. To specifically target BB, embryos were co-treated with isoproterenol, a ß-adrenergic agonist that stimulates the zfPMR, and the inhibition of isoproterenol-induced response was used as a biomarker of BB exposure. Our results reveal that the inhibition of isoproterenol-stimulated zfPMRs can be used as a biosensor capable of detecting BB in the parts-per-billion to parts-per-trillion in water samples, including diluted MWWE. The method developed detects BB in spite of the presence of other neuroactive compounds in water samples. This systems level approach of rapid screening for BB effects provides the most promising evidence to date that behavioral neuromodulation can be potentially applied for environmental effects monitoring of pharmaceuticals.

Nonlinear mixed-modelling discriminates the effect of chemicals and their mixtures on zebrafish behavior.

Zebrafish (Danio rerio) early-life stage behavior has the potential for high-throughput screening of neurotoxic environmental contaminants. However, zebrafish embryo and larval behavioral assessments typically utilize linear analyses of mean activity that may not capture the complexity of the behavioral response. Here we tested the hypothesis that nonlinear mixed-modelling of zebrafish embryo and larval behavior provides a better assessment of the impact of chemicals and their mixtures. We demonstrate that zebrafish embryo photomotor responses (PMRs) and larval light/dark locomotor activities can be fit by asymmetric Lorentzian and Ricker-beta functions, respectively, which estimate the magnitude of activity (e.g., maximum and total activities) and temporal aspects (e.g., duration of the responses and its excitatory periods) characterizing early life-stage zebrafish behavior. We exposed zebrafish embryos and larvae to neuroactive chemicals, including isoproterenol, serotonin, and ethanol, as well as their mixtures, to assess the feasibility of using the nonlinear mixed-modelling to assess behavioral modulation. Exposure to chemicals led to distinct effects on specific behavioral characteristics, and interactive effects on temporal characteristics of the behavioral response that were overlooked by the linear analyses of mean activity. Overall, nonlinear mixed-modelling is a more comprehensive approach for screening the impact of chemicals and chemical mixtures on zebrafish behavior.

Lifelong Exposure to PCBs in the Remote Norwegian Arctic Disrupts the Plasma Stress Metabolome in Arctic Charr.

Lake Ellasjøen on the remote Norwegian island of Bjørnøya is populated by Arctic charr (Salvelinus alpinus) having 20-fold higher body burdens of polychlorinated biphenyls (PCB) compared to charr from the neighboring Lake Laksvatn. This provides a natural setting to test the hypothesis that lifelong exposure to PCBs compromises the energy metabolism in this northernmost living salmonid. To test this, blood was sampled from charr from both lakes immediately after capture and following a 1 h handling and confinement stressor to assess possible differences in their energy metabolism and energy substrate mobilization, respectively. The plasma metabolome of charr was assessed by metabolite detection/separation with LC-MS. Plasma metabolite profiles revealed differences in key pathways involved in amino acid metabolism between charr from each lake, underscoring an impact of PCBs on energy metabolism in Arctic charr residing in Lake Ellasjøen. Subjecting charr from either lake to an acute stressor altered the plasma metabolite profiles and revealed distinct stress metabolome in Lake Ellasjøen charr, suggesting a reduced metabolic capacity. Taken together, lifelong exposure to PCBs in Ellasjøen charr disrupts the plasma metabolome, and may impair the adaptive metabolic response to stressors, leading to a reduced fitness.

Testing Local Adaptation in Five Populations of Hyalella azteca in Northern Alberta's Oil Sands Region.

Canada's oil sands hold the third largest petroleum reserves worldwide and have experienced rapid economic growth. The oil sands region provides an ideal location for studying local adaptations through reciprocal transplant (RT) because populations within the region have been historically exposed to naturally occurring bitumen. Our objectives were to (1) determine if Hyalella azteca from habitats within the oil sands region exhibited increased tolerance to constituents associated with industrial bitumen extraction compared with H. azteca from habitats outside the region; and (2) determine if any observed tolerance was attributable to local adaptation. Five populations of H. azteca were reciprocally transplanted from reclaimed and reference wetlands: four from local wetlands plus one naïve laboratory population. Survival, toxicity, and behaviour were measured before and after the RT period. Survival varied by population and site. These results show that the differences in responses among populations are likely not attributable to local adaptation and that laboratory populations of H. azteca provide ecologically relevant results when tested in the field.

Behavioural alterations from exposure to Cu, phenanthrene, and Cu-phenanthrene mixtures: linking behaviour to acute toxic mechanisms in the aquatic amphipod, Hyalella azteca.

Phenanthrene (PHE) and Cu are two contaminants commonly co-occurring in marine and freshwater environments. Mixtures of PHE and Cu have been reported to induce more-than-additive lethality in the amphipod, Hyalella azteca, a keystone aquatic invertebrate, yet little is understood regarding the interactive toxic mechanisms that mediate more-than-additive toxicity. Understanding the interactions among toxic mechanisms among Cu and PHE will allow for better predictive power in assessing the ecological risks of Cu-PHE mixtures in aquatic environments. Here we use behavioural impairment to help understand the toxic mechanisms of Cu, PHE, and Cu-PHE mixture toxicity in the aquatic amphipod crustacean, Hyalella azteca. Our principal objective was to link alterations in activity and ventilation with respiratory rates, oxidative stress, and neurotoxicity in adult H. azteca. Adult amphipods were used for all toxicity tests. Amphipods were tested at sublethal exposures of 91.8- and 195-µgL(-1) Cu and PHE, respectively, and a Cu-PHE mixture at the same concentrations for 24h. Neurotoxicity was measured as acetylcholinesterase (AChE) activity, where malathion was used as a positive control. Oxidative stress was measured as reactive oxygen species (ROS) production. Phenanthrene-exposed amphipods exhibited severe behavioural impairment, being hyperstimulated to the extent that they were incapable of coordinating muscle movements. In addition, respiration and AChE activity in PHE-exposed amphipods were increased and reduced by 51% and 23% respectively. However, ROS did not increase following exposure to phenanthrene. In contrast, Cu had no effect on amphipod behaviour, respiration or AChE activity, but did lead to an increase in ROS. However, co-exposure to Cu antagonized the PHE-induced reduction in ventilation and negated any increase in respiration. The results suggest that PHE acts like an organophosphate pesticide (e.g., malathion) in H. azteca following 24h sublethal exposures, and that AChE inhibition is the likely mechanism by which PHE alters H. azteca behaviour. However, interactive aspects of neurotoxicity do not account for the previously observed more-than-additive mortality in H. azteca following exposure to Cu-PHE mixtures.

Metal-Polycyclic Aromatic Hydrocarbon Mixture Toxicity in Hyalella azteca. 2. Metal Accumulation and Oxidative Stress as Interactive Co-toxic Mechanisms.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) are commonly found in aquatic environments. Emerging reports have identified that more-than-additive mortality is common in metal-PAH mixtures. Individual aspects of PAH toxicity suggest they may alter the accumulation of metals and enhance metal-derived reactive oxygen species (ROS). Redox-active metals (e.g., Cu and Ni) are also capable of enhancing the redox cycling of PAHs. Accordingly, we explored the mutual effects redox-active metals and PAHs have on oxidative stress, and the potential for PAHs to alter the accumulation and/or homeostasis of metals in juvenile Hyalella azteca. Amphipods were exposed to binary mixtures of Cu, Cd, Ni, or V, with either phenanthrene (PHE) or phenanthrenequinone (PHQ). Mixture of Cu with either PAH produced striking more-than-additive mortality, whereas all other mixtures amounted to strictly additive mortality following 18-h exposures. We found no evidence to suggest that interactive effects on ROS production were involved in the more-than-additive mortality of Cu-PHE and Cu-PHQ mixtures. However, PHQ increased the tissue concentration of Cu in juvenile H. azteca, providing a potential mechanism for the observed more-than-additive mortality.

Metal-Polycyclic Aromatic Hydrocarbon Mixture Toxicity in Hyalella azteca. 1. Response Surfaces and Isoboles To Measure Non-additive Mixture Toxicity and Ecological Risk.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their potential to produce non-additive toxicity (i.e., antagonism or potentiation). A review of the lethality of metal-PAH mixtures in aquatic biota revealed that more-than-additive lethality is as common as strictly additive effects. Approaches to ecological risk assessment do not consider non-additive toxicity of metal-PAH mixtures. Forty-eight-hour water-only binary mixture toxicity experiments were conducted to determine the additive toxic nature of mixtures of Cu, Cd, V, or Ni with phenanthrene (PHE) or phenanthrenequinone (PHQ) using the aquatic amphipod Hyalella azteca. In cases where more-than-additive toxicity was observed, we calculated the possible mortality rates at Canada's environmental water quality guideline concentrations. We used a three-dimensional response surface isobole model-based approach to compare the observed co-toxicity in juvenile amphipods to predicted outcomes based on concentration addition or effects addition mixtures models. More-than-additive lethality was observed for all Cu-PHE, Cu-PHQ, and several Cd-PHE, Cd-PHQ, and Ni-PHE mixtures. Our analysis predicts Cu-PHE, Cu-PHQ, Cd-PHE, and Cd-PHQ mixtures at the Canadian Water Quality Guideline concentrations would produce 7.5%, 3.7%, 4.4% and 1.4% mortality, respectively.

Metal-PAH mixtures in the aquatic environment: a review of co-toxic mechanisms leading to more-than-additive outcomes.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their combined toxicities. Emerging reports investigating the additive mortality in metal-PAH mixtures have indicated that more-than-additive effects are equally as common as strictly-additive effects, raising concern for ecological risk assessment typically based on the summation of individual toxicities. Moreover, the current separation of focus between in vivo and in vitro studies, and fine- and coarse-scale endpoints, creates uncertainty regarding the mechanisms of co-toxicity involved in more-than-additive effects on whole organisms. Drawing from literature on metal and PAH toxicity in bacteria, protozoa, invertebrates, fish, and mammalian models, this review outlines several key mechanistic interactions likely to promote more-than-additive toxicity in metal-PAH mixtures. Namely, the deleterious effects of PAHs on membrane integrity and permeability to metals, the potential for metal-PAH complexation, the inhibitory nature of metals to the detoxification of PAHs via the cytochrome P450 pathway, the inhibitory nature of PAHs towards the detoxification of metals via metallothionein, and the potentiated production of reactive oxygenated species (ROS) in certain metal (e.g. Cu) and PAH (e.g., phenanthrenequinone) mixtures. Moreover, the mutual inhibition of detoxification suggests the possibility of positive feedback among these mechanisms. The individual toxicities and interactive aspects of contaminant transport, detoxification, and the production of ROS are herein discussed.