Lack of genotoxicity potential and safety assessment of 4-(2-hydroxyethyl) morpholine present as an impurity in pharmaceuticals and nutritional supplements.

4-(2-Hydroxyethyl) morpholine (HEM) is widely used as a building block of macromolecules in the manufacture of pharmaceuticals and dietary supplements and could remain as an impurity in the finished products. An evaluation of HEM was conducted to identify endpoints that could be used to determine the point-of-departure (POD) for use in assessing the potential risk from exposure to HEM. No oral repeated dose toxicological studies of appropriate duration were found for HEM. Therefore, suitable analogue(s) were identified. Although oral repeated dose studies were available for the analogues, the studies were not of sufficient duration for use in the assignment of a POD for risk evaluation. Accordingly, the Threshold of Toxicological Concern (TTC) approach, which proposes that a de minimis value can be derived to qualitatively assess risk, was considered for HEM. To determine the appropriate TTC approach (genotoxic or non-genotoxic), the genotoxicity of HEM and its analogues were evaluated. The weight of the evidence indicated that HEM, and the appropriate analogues, are not genotoxic. Considering the chemical structure of HEM, the non-genotoxic Cramer class III TTC value of 1.5 µg/kg bw/day was determined to be appropriate for use in safety assessment of HEM as an impurity in products intended for human consumption.

Combined Developmental Toxicity of the Pesticides Difenoconazole and Dimethomorph on Embryonic Zebrafish.

Difenoconazole (DIF) and dimethomorph (DIM) are widely used pesticides frequently detected together in environmental samples, so the deleterious effects of combined exposure warrant detailed examination. In this study, the individual and combined effects of DIM and DIF on conventional developmental parameters (hatching rate, deformity rate, lethality) and gene expression were measured in embryonic zebrafish. Both DIF and DIM interfered with normal zebrafish embryo development, and the most sensitive toxicity index for both was 96 h post-fertilization (hpf) deformity rate (BMDL10 values of 0.30 and 1.10 mg/L, respectively). The combination of DIF and DIM had mainly synergistic deleterious effects on 96 hpf deformity and mortality rates. Transcriptome analysis showed that these compounds markedly downregulated expression of mcm family genes, cdk1, and cdc20, thereby potentially disrupting DNA replication and cell cycle progression. Enhanced surveillance for this pesticide combination is recommended as simultaneous environmental exposure may be substantially more harmful than exposure to either compound alone.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

Prenatal cannabinoid exposure alters the ovarian reserve in adult offspring of rats.

In animals, research in the past two decades has demonstrated the strong involvement of the endocannabinoid system (ECS) in numerous steps of the reproductive process, including ovarian physiology. Reproductive lifespan is closely related to the number of nongrowing ovarian follicles, called ovarian reserve (OR), which is definitively established during foetal life. Thus, OR damage may lead to poor reproductive outcomes and a shortened reproductive lifespan. We investigated whether prenatal ECS modulation had an effect on the OR at different ages in the rat offspring. Four groups of gestating female rats (F0) were exposed to the CB1-/CB2-receptor agonist WIN55212 (0.5 mg/kg), the CB1R inverse agonist SR141716 (3 mg/kg) or Δ9THC (5 mg/kg) and were compared to negative control groups. OR was histologically assessed at different postnatal timepoints (F1 individuals): postnatal day (PND) 6, PND40 and PND90. At PND6, prenatal exposure had no effect on OR. In the young adult group (PND90) exposed during gestation to WIN55212, we observed a CB1R-mediated delayed OR decrease, which was reversed by prenatal CB1R blockade by SR141716. Conversely, after prenatal SR141716 exposure, we observed higher OR counts at PND90. RT-PCR experiments also showed that prenatal ECS modulation perturbed the mRNA levels of ECS enzymes and OR regulation genes. Our findings support the role of the ECS in OR regulation during the foetal life of rats and highlight the need for further studies to elucidate its precise role in OR physiology.

Cannabinoids induce latent sensitization in a preclinical model of medication overuse headache.

AIM: Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache. METHODS: Female Sprague Dawley rats received graded intraperitoneal doses of WIN55,212-2 or Δ-9-tetrahydrocannabinol (Δ-9-THC). Antinociception (tail-flick test), catalepsy and hypomotility (open field test) and impairment of motor function (rotarod test) were assessed to establish effective dosing. Rats were then treated twice daily with equianalgesic doses of WIN55,212-2 or Δ-9-THC, or vehicle, for 7 days and cutaneous tactile sensory thresholds were evaluated during and three weeks following drug discontinuation. Rats then received a one-hour period of bright light stress (BLS) on two consecutive days and tactile sensory thresholds were re-assessed. RESULTS: WIN55,212-2 and Δ-9-THC produced antinociception as well as hypomotility, catalepsy and motor impairment. Repeated administration of WIN55,212-2 and Δ-9-THC induced generalized periorbital and hindpaw allodynia that resolved within 3 weeks after discontinuation of drug. Two episodes of BLS produced delayed and long-lasting periorbital and hindpaw allodynia selectively in rats previously treated with WIN55,212-2, and Δ-9-THC. INTERPRETATION: Cannabinoid receptor agonists including Δ-9-THC produce a state of latent sensitization characterized by increased sensitivity to stress, a presumed migraine trigger. Overuse of cannabinoids including cannabis may increase the risk of medication overuse headache in vulnerable individuals.

Inhibition of Ku70 in a high-glucose environment aggravates bupivacaine-induced dorsal root ganglion neurotoxicity.

BACKGROUND: Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. METHODS: We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. RESULTS: Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. CONCLUSION: These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.

Sex-specific behavioural deficits induced at early life by prenatal exposure to the cannabinoid receptor agonist WIN55, 212-2 depend on mGlu5 receptor signalling.

BACKGROUND AND PURPOSE: Marijuana is the illicit drug most commonly used among pregnant and breastfeeding women. Different studies reported long-term adverse effects induced by in utero exposure to the main component of marijuana, Δ9 -tetrahydrocannabinol (THC), both in rodents and in humans. However, little is known about any potential sex-dependent effects of marijuana consumption during pregnancy on newborns at early developmental ages. EXPERIMENTAL APPROACH: We studied the effects of prenatal exposure to the cannabinoid receptor agonist WIN55,212-2 (WIN; 0.5 mg·kg-1 from GD5 to GD20) on the emotional reactivity and cognitive performance of male and female rat offspring from infancy through adolescence and tested the role of mGlu5 receptor signalling in the observed effects. KEY RESULTS: Prenatally WIN-exposed male infant pups emitted less isolation-induced ultrasonic vocalizations compared with male control pups, when separated from the dam and siblings and showed increased locomotor activity while females were spared. These effects were normalized when male pups were treated with the positive allosteric modulator of mGlu5 receptor CDPPB. When tested at the prepubertal and pubertal periods, WIN-prenatally exposed rats of both sexes did not show any difference in social play behaviour, anxiety and temporal order memory. CONCLUSIONS AND IMPLICATIONS: We reveal a previously undisclosed sexual divergence in the consequences of fetal cannabinoids on newborns at early developmental ages, which is dependent on mGlu5 receptor signalling. These results provide new impetus for the urgent need to investigate the functional and behavioural substrates of prenatal cannabinoid exposure in both the male offspring and the female offspring.

Evaluation of preclinical safety profile of SPH3127, a direct renin inhibitor, after 28-day repeated oral administration in Sprague-Dawley rats and cynomolgus monkeys.

SPH3127, a newly developed oral nonpeptide direct renin inhibitor with good tolerance and favorable ADME (absorption distribution metabolism excretion) properties in preclinical species, is now being evaluated in phase Ι clinical trial. In this work, the subchronic toxicity of SPH3127 in Sprague-Dawley rats and cynomolgus monkeys has been characterized. Rats and monkeys received SPH3127 orally (30, 300, 900 and 20, 100, 450 mg/kg/day, respectively) on a consecutive daily dosing schedule for 28 days followed by a 28-days recovery period for one third of the animals. The adverse effects of SPH3127 on rats and monkeys mainly included kidney and cardiovascular toxicity, which were consistent with pharmacologic perturbations of physiologic processes associated with the intended molecular targets for this class of renin signaling inhibitors. Moderate liver weight increases accompanied by CYP3A induction were seen in 300 and 900 mg/kg/day rats but not in monkeys or in vitro human hepatocytes. One 450 mg/kg/day monkey died early at day 23 with apparent myelosuppression characterized by atrophy of thymus and spleen, and the relevance to the action of SPH3127 remained unclear. Most of the treatment-induced effects were reversible upon discontinuation of treatment. The no-observed-adverse-effect level (NOAEL) of SPH3127 was determined to be 30 mg/kg/day for Sprague-Dawley rats and 20 mg/kg/day for cynomolgus monkeys based on the kidney and cardiovascular changes found at mid- and high-dose animals.

A structure optimized fluorescent probe for highly sensitive monitoring drug induced lysosomal pH value changes.

Lysosomes generally maintain the weak acidic microenvironment, to ensure highly efficient activity and functions of hydrolytic enzymes and proteins. Aberrations of the lysosomal pH may result in cellular functional changes and influence human physiology, possibly causing serious diseases. Small-molecular fluorescent probes based imaging techniques capable of providing information on target locations are considerably appreciated. Herein, by reducing the size of the typical lysosome targetable group 4-(2-aminoethyl) morpholine, we rationally designed a rhodamine analogue probe Ly-HN2AM with N-Aminomorpholine as the ring-closed switch and the lysosome targetable moiety for visualizing lysosomal pH changes. With the benefit of constructing multi-pentacyclic intramolecular hydrogen bond when binding with the H+, Ly-HN2AM gives a highly sensitive response towards pH values ranging from 4.79 to 6.07, with a remarkable higher pKa 5.35 over the typical 4-(2-aminoethyl) morpholine modified probes. The new probe was successfully applied to visualize pH value changes in lysosome-associated physiological and pathological processes with excellent photostability and low cytotoxicity, indicating the potential applications of lysosome specific bioimaging.

Memory deficits induced by chronic cannabinoid exposure are prevented by adenosine A2AR receptor antagonism.

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212-2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB1R) and cancelled by concomitant administration of adenosine A2A receptor (A2AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by A2AR antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212-2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212-2 (300 nM), an effect partially rescued by the A2AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A2AR or CB1R binding in the hippocampus and in the prefrontal cortex. These results, showing that A2AR antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A2ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.

Novel quinazolin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.

Synthetic cannabinoids and endometrial stromal cell fate: Dissimilar effects of JWH-122, UR-144 and WIN55,212-2.

The emergence of synthetic cannabinoids (SCBs) as drugs of abuse in readily available "Spice" smoking blends has exposed users to much more potent cannabinoids than the phytocannabinoids present in Cannabis sativa L. Increasing reports of adverse reactions are emerging in the clinical literature. SCBs may disrupt the endocannabinoid signalling, which has been shown to be crucial within human endometrium remodelling process. Within this study, a telomerase-immortalised human endometrial stromal cell line (St-T1b) and primary human decidual fibroblasts (HdF) were used to determine the impact of SCBs JWH-122, UR-144 and WIN55,212-2 (WIN) on endometrial stromal cells. Our findings indicate that JWH-122 and UR-144 (0.01-25 µM) induce prompt ROS/RNS formation and endoplasmic reticulum (ER) stress without reduction in cell viability. Disturbances in the normal functions of the ER lead to cell stress response, which is after compensated with the increase in reduced/oxidized glutathione ratio (GSH/GSSG). Instead, WIN induces ER stress, mitochondrial dysfunction and apoptotic cell death. The addition of the CB1 antagonist AM281 significantly reduces the effects on cell viability, suggesting that CB1 plays a key role in WIN-induced apoptosis. Collectively, our data suggests that SCBs have dissimilar effects on human endometrial stromal cells and, thus, may impact human reproductive function through distinct mechanisms that are crucial for the understanding of the pathophysiological outcomes from its abuse.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro.

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC50 values of 5 µM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K+-elicited [Ca2+]c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca2+ entry and the uncoupling of the Ca2+-dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death.

Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models.

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

Effect of Cannabinoid Receptor Agonists on Isolated Rat Atria.

Cannabinoid CB2 receptor agonists are under investigation for clinical use. At the same time, synthetic cannabinoids have been implicated in a number of deaths. One cause of death is thought to be cardiac arrest subsequent to extreme tachycardia. Central mechanisms are thought to play a role in this, with CB1 but not CB2 receptors thought to mediate central effects. However, the direct effects of cannabinoids on the heart are less well understood. We therefore tested the effects of cannabinoids on isolated rat atria to test whether activation of myocardial CB1 and CB2 receptors could contribute to tachycardia. Although we found a moderate effect that can be attributed to CB1 receptors, we did not find any evidence for chronotropic effects by a CB2 receptor activation. Our results indicate that cannabinoid cardiotoxicity may partially involve CB1 receptors in the myocardium, and that CB2 receptor agonists are unlikely to have significant effects on the heart.

A photostable fluorescent probe for long-time imagining of lysosome in cell and nematode.

Lysosome fluorescent imaging has been widely used in the field of biological staining and diagnostics, which plays a key role in understanding intracellular metabolism and various physiological processes. However, for most currently used small-molecule lysotrackers, the photostability is often unsatisfactory when used for long-term and real-time visualization of lysosomal dynamics. Herein, we reported a new lysosome-targetable photostable fluorescent probe (i.e. MPL-NPA), and results showed that MPL-NAP possesses superior photostability, appreciable tolerance to pH change, low cytotoxicity and high lysosome targeting ability. These findings confirm that MPL-NAP is a well-suited imaging agent for targeting lysosome and enables long-term and real-time monitor of lysosome morphological changes under physiological processes.

Chronic, intermittent treatment with a cannabinoid receptor agonist impairs recognition memory and brain network functional connectivity.

Elucidating how cannabinoids affect brain function is instrumental for the development of therapeutic tools aiming to mitigate 'on target' side effects of cannabinoid-based therapies. A single treatment with the cannabinoid receptor agonist, WIN 55,212-2, disrupts recognition memory in mice. Here, we evaluate how prolonged, intermittent (30 days) exposure to WIN 55,212-2 (1 mg/kg) alters recognition memory and impacts on brain metabolism and functional connectivity. We show that chronic, intermittent treatment with WIN 55,212-2 disrupts recognition memory (Novel Object Recognition Test) without affecting locomotion and anxiety-like behaviour (Open Field and Elevated Plus Maze). Through 14 C-2-deoxyglucose functional brain imaging we show that chronic, intermittent WIN 55,212-2 exposure induces hypometabolism in the hippocampal dorsal subiculum and in the mediodorsal nucleus of the thalamus, two brain regions directly involved in recognition memory. In addition, WIN 55,212-2 exposure induces hypometabolism in the habenula with a contrasting hypermetabolism in the globus pallidus. Through the application of the Partial Least Squares Regression (PLSR) algorithm to the brain imaging data, we observed that prolonged WIN 55,212-2 administration alters functional connectivity in brain networks that underlie recognition memory, including that between the hippocampus and prefrontal cortex, the thalamus and prefrontal cortex, and between the hippocampus and the perirhinal cortex. In addition, our results support disturbed lateral habenula and serotonin system functional connectivity following WIN 55,212-2 exposure. Overall, this study provides new insight into the functional mechanisms underlying the impact of chronic cannabinoid exposure on memory and highlights the serotonin system as a particularly vulnerable target.

Impacts of temperature, morpholine, and chronic radiation on the embryonic development of round whitefish (Prosopium cylindraceum).

During incubation, round whitefish embryos may experience fluctuating or elevated temperatures from natural (e.g., seasonal temperature changes) and/or anthropogenic sources. Anthropogenic sources like once-through cooling discharges from nuclear power plants can also expose embryos to chemicals (e.g., morpholine) and/or radiation. To examine the effects of these potential stressors on embryogenesis, round whitefish were incubated under fluctuating or constant temperatures, with morpholine or 137 Cs gamma rays. We report the percentage of prehatch and posthatch mortality, developmental rate, hatch dynamics, and morphometrics at 4 development stages. Embryos reared at constant temperatures had delayed developmental stage onset and median hatch, higher mortality at constant 8 °C, and lower mortality at ≤5 °C, compared with embryos reared under seasonal temperature regimes. Embryos incubated with ≥500 mg L-1 morpholine (>200× regulatory limits) had advanced hatch, reduced body size, and increased prehatch (100% at 1000 mg L-1 ) and posthatch (≈95% at 500 mg L-1 ) mortality compared with controls. Relative to controls, embryos irradiated with ≥0.16 mGy/d had larger body mass early in development, and all irradiated embryos had decreased posthatch mortality; the lowest dose was >300× discharge limits. Our study suggests that fluctuating or elevated temperatures and high-dose morpholine can alter development rate, hatch dynamics, and growth, and/or increase mortality compared with embryos reared at constant temperatures of ≤5 °C; conversely, low-dose irradiation had transient developmental effects but may benefit early posthatch survival. Environ Toxicol Chem 2018;37:2593-2608. © 2018 SETAC.