Assessing the Toxicity of Benzotriazole Ultraviolet Stabilizers to Fishes: Insights into Aryl Hydrocarbon Receptor-Mediated Effects.

Benzotriazole ultraviolet stabilizers (BUVSs) are chemicals used to mitigate UV-induced damage to manufactured goods. Their presence in aquatic environments and biota raises concerns, as certain BUVSs activate the aryl hydrocarbon receptor (AhR), which is linked to adverse effects in fish. However, potencies of BUVSs as AhR agonists and species sensitivities to AhR activation are poorly understood. This study evaluated the toxicity of three BUVSs using embryotoxicity assays. Zebrafish (Danio rerio) embryos exposed to BUVSs by microinjection suffered dose-dependent increases in mortality, with LD50 values of 4772, 11 608, and 56 292 ng/g-egg for UV-P, UV-9, and UV-090, respectively. The potencies and species sensitivities to AhR2 activation by BUVSs were assessed using a luciferase reporter gene assay with COS-7 cells transfected with the AhR2 of zebrafish and eight other fishes. The rank order of potency for activation of the AhR2 from all nine species was UV-P > UV-9 > UV-090. However, AhR2s among species differed in sensitivities to activation by up to 100-fold. An approximate reversed rank order of species sensitivity was observed compared to the rank order of sensitivity to 2,3,7,8-tetrachlorodibenzo[p]dioxin, the prototypical AhR agonist. Despite this, a pre-existing quantitative adverse outcome pathway linking AhR activation to embryo lethality could predict embryotoxicities of BUVSs in zebrafish.

Endogenous AhR agonist FICZ accumulates in rainbow trout (Oncorhynchus mykiss) alevins exposed to a mixture of two PAHs, retene and fluoranthene.

Multiple studies have reported synergized toxicity of PAH mixtures in developing fish larvae relative to the additive effect of the components. From a toxicological perspective, multiple mechanisms are known to contribute to synergism, such as altered toxicodynamics and kinetics, as well as increased oxidative stress. An understudied contributor to synergism is the accumulation of endogenous metabolites, for example: the aryl hydrocarbon receptor 2 (AhR2) agonist and tryptophan metabolite 6-Formylindolo(3,2-b)carbazole (FICZ). Fish larvae exposed to FICZ, alongside knock-down of cytochrome p450 (cyp1a), has been reported to induced symptoms of toxicity similar to those observed following exposure to PAHs or the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we explored if FICZ accumulates in newly hatched rainbow trout alevins (Oncorhynchus mykiss) exposed to two PAHs with different properties: retene (potent AhR2 agonist) and fluoranthene (weak AhR2 agonist and Cyp1a inhibitor), either alone or as a binary mixture for 3 and 7 days. We found that exposure to the mixture resulted in accumulation of endogenous FICZ, synergized the blue sac disease index (BSD), and altered the body burden profiles of the PAHs, when compared to the alevins exposed to the individual components. It is thus very plausible that accumulation of endogenously derived FICZ contributed to the synergized BSD index and toxicity in exposed alevins. Accumulation of endogenously derived FICZ is a novel finding that extends our general understanding on PAHs toxicity in developing fish larvae, while at the same time highlighting why environmental risk assessment of PAHs should not be based solely results from the assessment of individual compounds.

Platelet adhesion in type 2 diabetes: impact of plasma albumin and mean platelet volume.

BACKGROUND: Altered mean platelet volume (MPV) and plasma albumin has been reported in type 2 diabetes (T2D). MPV is suggested to predict cardiovascular risk but there is a lack of evidence for associations between MPV and platelet adhesion. Plasma albumin and magnesium are other factors reported to influence thrombotic risk. The objectives of this study were to assess the association between platelet adhesion and plasma factors with a potential role to affect platelet activation. METHODS: Blood was collected from 60 T2D patients and 60 healthy controls. Platelet adhesion to different protein surfaces induced by various soluble activators were measured in microplates. MPV, albumin and magnesium were analysed together with additional routine tests. RESULTS: Despite normal levels, plasma albumin significantly correlated with adhesion of T2D platelets but not with controls. There was a significant association between MPV and platelet adhesion in both groups, but association was smaller in T2D. Levels of glucose, HbA1c or magnesium did not correlate with platelet adhesion. CONCLUSIONS: Plasma albumin was associated with platelet adhesion in T2D suggesting that albumin may be a factor to consider upon cardiovascular risk assessment. MPV was more associated with the level of platelet adhesion in healthy individuals than in well-controlled T2D patients.

A new low-dose multi-phase trauma CT protocol and its impact on diagnostic assessment and radiation dose in multi-trauma patients.

PURPOSE: Computed tomography (CT) examinations, often using high-radiation dosages, are increasingly used in the acute management of polytrauma patients. This study compares a low-dose polytrauma multi-phase whole-body CT (WBCT) protocol on a latest generation of 16-cm detector 258-slice multi-detector CT (MDCT) scanner with advanced dose reduction techniques to a single-phase polytrauma WBCT protocol on a 64-slice MDCT scanner. METHODS: Between March and September 2015, 109 polytrauma patients (group A) underwent acute WBCT with a low-dose multi-phase WBCT protocol on a 258-slice MDCT whereas 110 polytrauma patients (group B) underwent single-phase trauma CT on a 64-slice MDCT. The diagnostic accuracy to trauma-related injuries, radiation dose, quantitative and semiquantitative image quality parameters, subjective image quality scorings, and workflow time parameters were compared. RESULTS: In group A, statistically significantly more arterial injuries (p = 0.04) and arterial dissections (p = 0.002) were detected. In group A, the mean (±SD) dose length product value was 1681 ± 183 mGy*cm and markedly lower when compared to group B (p < 0.001). The SDs of the mean Houndsfield unit values of the brain, liver, and abdominal aorta were lower in group A (p < 0.001). Mean signal-to-noise ratios (SNRs) for the brain, liver, and abdominal aorta were significantly higher in group A (p < 0.001). Group A had significantly higher image quality scores for all analyzed anatomical locations (p < 0.02). However, the mean time from patient registration until completion of examination was significantly longer for group A (p < 0.001). CONCLUSIONS: The low-dose multi-phase CT protocol improves diagnostic accuracy and image quality at markedly reduced radiation. However, due to technical complexities and surplus electronic data provided by the newer low-dose technique, examination time increases, which reduces workflow in acute emergency situations.

Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

Embryonic Exposure to Benzotriazole Ultraviolet Stabilizer 327 Alters Behavior of Rainbow Trout Alevin.

Benzotriazole ultraviolet (UV) stabilizers (BUVSs) are used in great quantities during industrial production of a variety of consumer and industrial goods. As a result of leaching and spill, BUVSs are detectable ubiquitously in the environment. As of May 2023, citing concerns related to bioaccumulation, biomagnification, and environmental persistence, (B)UV(S)-328 was recommended to be listed under Annex A of the Stockholm Convention on Persistent Organic Pollutants. However, a phaseout of UV-328 could result in a regrettable substitution because the replacement chemical(s) could cause similar or unpredicted toxicity in vivo, relative to UV-328. Therefore, the influence of UV-327, a potential replacement of UV-328, was investigated with respect to early life development of newly fertilized rainbow trout embryos (Oncorhynchus mykiss), microinjected with environmentally relevant concentrations of UV-327. Developmental parameters (standard length), energy consumption (yolk area), heart function, blue sac disease, mortality, and behavior were investigated. Alevins at 14 days posthatching, exposed to 107 ng UV-327 g-1 egg, presented significant signs of hyperactivity; they moved on average 1.8-fold the distance and at 1.5-fold the velocity of controls. Although a substantial reduction in body burden of UV-327 was observed at hatching, it is postulated that UV-327, due to its lipophilic properties, interfered with neurological development and signaling from the onset of neurogenesis. If these results hold true across multiple taxa and species, a potential contributor to neurodevelopmental disorders might have been identified. These findings suggest that UV-327 poses an unknown hazard to rainbow trout embryos and alevins, rendering UV-327 a potential regrettable substitution to UV-328. However, a qualified statement on a regrettable substitution requires a comparative investigation on the teratogenic effects between the two BUVSs. Environ Toxicol Chem 2024;43:762-771. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Far from Their Origins: A Transcriptomic Investigation on How 2,4-Di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) Phenol Affects Rainbow Trout Alevins.

Benzotriazole ultraviolet stabilizers (BUVSs) are a group of widely used chemicals added to a variety of consumer (e.g., plastics) and industrial (e.g., metal coating) goods. Although detected globally as an environmentally persistent pollutant, BUVSs have received relatively little toxicological attention and only recently have been acknowledged to affect development and the endocrine system in vivo. In our previous study, altered behavior, indicative of potential neurotoxicity, was observed among rainbow trout alevins (day 14 posthatching) that were microinjected as embryos with a single environmentally relevant dose of 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) phenol (UV-327). In the present follow-up study, we performed whole-transcriptome profiling (RNA sequencing) of newly hatched alevins from the same batch. The primary aim was to identify biomarkers related to behavior and neurology. Dose-specifically, 1 to 176 differentially expressed genes (DEGs) were identified. In the group presenting altered behavior (273.4 ng g-1), 176 DEGs were identified, yet only a fraction was related to neurological functions, including water, calcium, and potassium homeostasis; acetylcholine transmission and signaling; as well insulin and energy metabolism. The second objective was to estimate the transcriptomic point of departure (tPOD) and assess if point estimate(s) are protective of altered behavior. A tPOD was established at 35 to 94 ng UV-327 g-1 egg, making this tPOD protective of behavioral alterations. Holistically, these transcriptomic alterations provide a foundation for future research on how BUVSs can influence rainbow trout alevin development, while providing support to the hypothesis that UV-327 can influence neurogenesis and subsequent behavioral endpoints. The exact structural and functional changes caused by embryonic exposure to UV-327 remain enigmatic and will require extensive investigation before being deciphered and understood toxicologically. Environ Toxicol Chem 2024;00:1-12. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Nanomolar concentrations of adrenaline induce platelet adhesion in vitro.

Adrenaline is a platelet activator having a resting plasma concentration of <1 nmol/l that increases to a few nmol/l during stress. However, most in vitro assays only detect effects of adrenaline in micromolar concentrations. This makes it difficult to estimate the relevance of in vitro data for the in vivo situation. The aim of this study was to investigate experimental conditions in vitro that could detect platelet effects of adrenaline in nanomolar concentrations. Platelet adhesion to albumin and collagen was evaluated with a static platelet adhesion assay. Our results show that 10 nmol/l adrenaline induced platelet adhesion to albumin in platelet-rich plasma (PRP) prepared at 140 × g, while 100 nmol/l was necessary in order to increase adhesion of platelets prepared at 220 × g. The mean platelet volume was increased after preparation at 140 × g, suggesting that large reactive platelets contributed to the increased adrenaline sensitivity. At optimal Mg(2+)-concentration, adhesion to collagen was increased by 10 nmol/l adrenaline irrespective of centrifugal force applied during PRP preparation. More specifically, we defined two populations where adhesion to collagen was increased by 10 nmol/l adrenaline either upon centrifugation at 140 × g but not 220 × g or vice versa. In some experiments, platelet adhesion to collagen was induced by 3 nmol/l adrenaline, which corresponds to concentrations achieved during stress in vivo. In summary, the static adhesion assay is able to detect platelet activating effects of adrenaline very close to physiological concentrations. This is rare for in vitro assays and motivates further research about adrenergic signalling in platelets.

Effects of Alkylation on Potency of Benz[a]anthracene for AhR2 Transactivation in Nine Species of Freshwater Fish.

Polycyclic aromatic hydrocarbons (PAHs) are naturally occurring or anthropogenic organic chemicals that can activate the aryl hydrocarbon receptor 2 (AhR2) and induce toxicity in fishes. Alkyl PAHs are more abundant than nonalkylated PAHs in certain environmental matrices and there is growing evidence that alkylation can increase potency, dependent on the position of alkylation. However, it is unknown if the effect of alkylation on potency is conserved across species. In addition, relatively little is known regarding the extent of interspecies variation in sensitivity to PAHs and alkyl PAHs. Therefore, objectives of the present study were to characterize potency of benz[a]anthracene (BAA) and three alkylated homologues representing different alkylation positions in nine phylogenetically diverse species of fish using a standardized in vitro AhR2 transactivation assay. BAA and each alkylated homologue activated the AhR2 in a concentration-dependent manner in each species. Position-dependent effects on potency were observed in every species, but these effects were not consistent across species. Interspecies variation in sensitivity to AhR2 activation by each PAH was observed and ranged by up to 561-fold. Alkylation both increased and decreased the range of interspecies variation and sensitivity, but the potency of each alkylated homologue relative to BAA ranged by less than an order of magnitude among species. These results represent an early step toward the consideration of alkylated homologues for more objective ecological risk assessments of PAHs to native fishes. Environ Toxicol Chem 2023;42:1575-1585. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Changes in cardiac proteome and metabolome following exposure to the PAHs retene and fluoranthene and their mixture in developing rainbow trout alevins.

Exposure to polycyclic aromatic hydrocarbons (PAHs) is known to affect developing organisms. Utilization of different omics-based technologies and approaches could therefore provide a base for the discovery of novel mechanisms of PAH induced development of toxicity. To this aim, we investigated how exposure towards two PAHs with different toxicity mechanisms: retene (an aryl hydrocarbon receptor 2 (Ahr2) agonist), and fluoranthene (a weak Ahr2 agonist and cytochrome P450 inhibitor (Cyp1a)), either alone or as a mixture, affected the cardiac proteome and metabolome in newly hatched rainbow trout alevins (Oncorhynchus mykiss). In total, we identified 65 and 82 differently expressed proteins (DEPs) across all treatments compared to control (DMSO) after 7 and 14 days of exposure. Exposure to fluoranthene altered the expression of 11 and 19 proteins, retene 29 and 23, while the mixture affected 44 and 82 DEPs by Days 7 and 14, respectively. In contrast, only 5 significantly affected metabolites were identified. Pathway over-representation analysis identified exposure-specific activation of phase II metabolic processes, which were accompanied with exposure-specific body burden profiles. The proteomic data highlights that exposure to the mixture increased oxidative stress, altered iron metabolism and impaired coagulation capacity. Additionally, depletion of several mini-chromosome maintenance components, in combination with depletion of several intermediate filaments and microtubules, among alevins exposed to the mixture, suggests compromised cellular integrity and reduced rate of mitosis, whereby affecting heart growth and development. Furthermore, the combination of proteomic and metabolomic data indicates altered energy metabolism, as per amino acid catabolism among mixture exposed alevins; plausibly compensatory mechanisms as to counteract reduced absorption and consumption of yolk. When considered as a whole, proteomic and metabolomic data, in relation to apical effects on the whole organism, provides additional insight into PAH toxicity and the effects of exposure on heart structure and molecular processes.

Exposure to retene, fluoranthene, and their binary mixture causes distinct transcriptomic and apical outcomes in rainbow trout (Oncorhynchus mykiss) yolk sac alevins.

Polycyclic aromatic hydrocarbons (PAHs) are widely spread environmental contaminants which affect developing organisms. It is known that improper activation of the aryl hydrocarbon receptor (AhR) by some PAHs contributes to toxicity, while other PAHs can disrupt cellular membrane function. The exact downstream mechanisms of AhR activation remain unresolved, especially with regard to cardiotoxicity. By exposing newly hatched rainbow trout alevins (Oncorhynchus mykiss) semi-statically to retene (32 µg l-1; AhR agonist), fluoranthene (50 µg l-1; weak AhR agonist and CYP1a inhibitor) and their binary mixture for 1, 3, 7 and 14 days, we aimed to uncover novel mechanisms of cardiotoxicity using a targeted microarray approach. At the end of the exposure, standard length, yolk area, blue sac disease (BSD) index and PAH body burden were measured, while the hearts were prepared for microarray analysis. Each exposure produced a unique toxicity profile. We observed that retene and the mixture, but not fluoranthene, significantly reduced growth by Day 14 compared to the control, while exposure to the mixture increased the BSD-index significantly from Day 3 onward. Body burden profiles were PAH-specific and correlated well with the exposure-specific upregulations of genes encoding for phase I and II enzymes. Exposure to the mixture over-represented pathways related to growth, amino acid and xenobiotic metabolism and oxidative stress responses. Alevins exposed to the individual PAHs displayed over-represented pathways involved in receptor signaling: retene downregulated genes with a role in G-protein signaling, while fluoranthene upregulated those involved in GABA signaling. Furthermore, exposure to retene and fluoranthene altered the expression of genes encoding for proteins involved in calcium- and potassium ion channels, which suggests affected heart structure and function. This study provides deeper understanding of the complexity of PAH toxicity and the necessity of investigating PAHs as mixtures and not as individual components.

Plasma levels of tissue inhibitor of metalloproteinase-1 in patients with type 1 diabetes mellitus associate with early diabetic neuropathy and nephropathy.

BACKGROUND: Tissue inhibitor of metalloproteinase-1 (TIMP-1) has been suggested as a marker for abnormal regulation of tissue remodelling in type 1 diabetes. Metalloproteinase-9 (MMP-9) has been associated with matrix turnover, and Neutrophil gelatinase associated lipocalin (NGAL) is a marker of tubular injury in diabetic nephropathy. The aim was to analyse these biomarkers to unmask early diabetic complications. METHODS: Thirty-three type 1 diabetes patients, aged 20-35 years, and disease duration 20 ± 5.3 years were included. Along with clinical examination, neurophysiological measurements, routine biochemistry, plasma concentrations of TIMP-1, MMP-9 and NGAL were determined with immunoenzymatic techniques. RESULTS: TIMP-1 correlated with abnormal unilateral and bilateral vibratory sense foot perception (r = -0.49 and r = -0.51, respectively), foot neuropathy impairment assessment score (NIA; r = -0.55), neuropathy symptom assessment score (r = 0.42), microalbuminuria (r = 0.50) and eGFR (r = -0.45). MMP-9 correlated with impaired foot NIA (r = 0.51). Multiple regression analysis showed an association for TIMP-1 (p = 0.004) with impaired neurophysiological examinations and renal dysfunction along with NGAL (p = 0.016 and p = 0.015 respectively). CONCLUSIONS: This study suggests that plasma levels of TIMP-1, MMP-9 and NGAL may serve as useful biomarkers in unravelling subclinical neuropathy and nephropathy in type 1 diabetes.

Pesticide-induced multigenerational effects on amphibian reproduction and metabolism.

Underlying drivers of species extinctions need to be better understood for effective conservation of biodiversity. Nearly half of all amphibian species are at risk of extinction, and pollution may be a significant threat as seasonal high-level agrochemical use overlaps with critical windows of larval development. The potential of environmental chemicals to reduce the fitness of future generations may have profound ecological and evolutionary implications. This study characterized effects of male developmental exposure to environmentally relevant concentrations of the anti-androgenic pesticide linuron over two generations of offspring in Xenopus tropicalis frogs. The adult male offspring of pesticide-exposed fathers (F1) showed reduced body size, decreased fertility, and signs of endocrine system disruption. Impacts were further propagated to the grand-offspring (F2), providing evidence of transgenerational effects in amphibians. The adult F2 males demonstrated increased weight and fat body palmitoleic-to-palmitic acid ratio, and decreased plasma glucose levels. The study provides important cross-species evidence of paternal epigenetic inheritance and pollutant-induced transgenerational toxicity, supporting a causal and complex role of environmental contamination in the ongoing species extinctions, particularly of amphibians.

Horse chestnut extract contracts bovine vessels and affects human platelet aggregation through 5-HT(2A) receptors: an in vitro study.

Extract from seeds and bark of horse chestnut (Aesculus hippocastanum L) is used as an herbal medicine against chronic venous insufficiency. The effect and mechanism of action on veins, arteries, and platelets are not fully understood. The aim of this study was to investigate the effects and mechanisms of action of horse chestnut on the contraction of bovine mesenteric veins and arteries, and human platelet aggregation. Contraction studies showed that horse chestnut extract dose-dependently contracted both veins and arteries, with the veins being the most sensitive. Contraction of both veins and arteries were significantly inhibited by the 5-HT(2A) receptor antagonist ketanserin. No effect on contraction was seen with the cyclooxygenase inhibitor indomethacin, the alpha(1) receptor antagonist prazosin or the angiotensin AT(1) receptor antagonist saralasin neither in veins nor arteries. ADP-induced human platelet aggregation was significantly reduced by horse chestnut. A further reduction was seen with the extract in the presence of ketanserin. In conclusion, horse chestnut contraction of both veins and arteries is, at least partly, mediated through 5-HT(2A) receptors. Human platelet aggregation is reduced by horse chestnut. The clinical importance of these findings concerning clinical use, possible adverse effects, and drug interactions remains to be investigated.

Enhanced platelet adhesion in essential thrombocythemia after in vitro activation.

OBJECTIVE: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by elevated platelet counts and increased risk of thrombosis. Ex vivo data suggest increased platelet reactivity in agreement with the increased thrombosis risk, while in vitro tests often detect decreased platelet activity. The present study aimed to investigate adhesion of ET-platelets in vitro, which is an aspect of platelet function that has been addressed in only a few studies on ET patients. METHODS: The study included 30 ET patients and 14 healthy controls. Platelet adhesion was measured with a static platelet adhesion assay. RESULTS: The main finding was that ET-platelets were more readily activated by adhesion-inducing stimuli in vitro than control platelets. This was particularly evident in elderly patients and when using multiple stimuli, such as surfaces of collagen or fibrinogen combined with addition of adenosine 5'-diphosphate or ristocetin. Such multiple stimuli resulted in adhesion above the control mean +2 standard deviations for approximately 50% of the patients. CONCLUSION: The results are in accordance with the concept of increased platelet activity in ET, but opposite to most other in vitro studies. We suggest that the conditions in the adhesion assay might mimic the in vivo situation regarding the presence of chronic platelet activation.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 2 - Proteomics and metabolomics.

Polycyclic aromatic hydrocarbons (PAHs) are global contaminants of concern. Despite several decades of research, their mechanisms of toxicity are not very well understood. Early life stages of fish are particularly sensitive with the developing cardiac tissue being a main target of PAHs toxicity. The mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons (PAHs) retene, pyrene and phenanthrene were explored in rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to sublethal doses of each individual PAH causing no detectable morphometric alterations. Changes in the cardiac proteome and metabolome were assessed after 7 or 14 days of exposure to each PAH. Phase I and II enzymes regulated by the aryl hydrocarbon receptor were significantly induced by all PAHs, with retene being the most potent compound. Retene significantly altered the level of several proteins involved in key cardiac functions such as muscle contraction, cellular tight junctions or calcium homeostasis. Those findings were quite consistent with previous reports regarding the effects of retene on the cardiac transcriptome. Significant changes in proteins linked to iron and heme metabolism were observed following exposure to pyrene. While phenanthrene also altered the levels of several proteins in the cardiac tissue, no clear mechanisms or pathways could be highlighted. Due to high variability between samples, very few significant changes were detected in the cardiac metabolome overall. Slight but significant changes were still observed for pyrene and phenanthrene, suggesting possible effects on several energetic or signaling pathways. This study shows that early exposure to different PAHs can alter the expression of key proteins involved in the cardiac function, which could potentially affect negatively the fitness of the larvae and later of the juvenile fish.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 1 - Transcriptomics.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants of concern that impact every sphere of the environment. Despite several decades of research, their mechanisms of toxicity are still poorly understood. This study explores the mechanisms of cardiotoxicity of the three widespread model PAHs retene, pyrene and phenanthrene in the rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to each individual compound at sublethal doses causing no significant increase in the prevalence of deformities. Changes in the cardiac transcriptome were assessed after 1, 3, 7 and 14 days of exposure using custom Salmo salar microarrays. The highest number of differentially expressed genes was observed after 1 or 3 days of exposure, and retene was the most potent compound in that regard. Over-representation analyses suggested that genes related to cardiac ion channels, calcium homeostasis and muscle contraction (actin binding, troponin and myosin complexes) were especially targeted by retene. Pyrene was also able to alter similar myosin-related genes, but at a different timing and in an opposite direction, suggesting compound-specific mechanisms of toxicity. Pyrene and to a lesser extent phenanthrene were altering key genes linked to the respiratory electron transport chain and to oxygen and iron metabolism. Overall, phenanthrene was not very potent in inducing changes in the cardiac transcriptome despite being apparently metabolized at a slower rate than retene and pyrene. The present study shows that exposure to different PAHs during the first few days of the swim-up stage can alter the expression of key genes involved into the cardiac development and function, which could potentially affect negatively the fitness of the larvae in the long term.

A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 µg/L)/linuron (9 or 45 µg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study.

BACKGROUND: Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. METHODS: With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. RESULTS: The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone. CONCLUSION: The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.

Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion.

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the alpha2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against alphaIIbbeta3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase alphaIIbbeta3-mediated platelet adhesion to albumin, dependent on alpha2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.