24-h Exposure Research Articles

Methylmalonic Acid Impairs Cell Respiration and Glutamate Uptake in C6 Rat Glioma Cells: Implications for Methylmalonic Acidemia.

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10mM) for 24 or 48h and cell metabolic viability, glucose consumption, and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell metabolic viability after 48-h period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-h exposure, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposure to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease. Methylmalonic acid compromises mitochondrial functioning leading to reduced ATP production and reduces glutamate uptake by C6 astroglial cells.

Multifunctional Silver Nanoparticles Based on Chitosan: Antibacterial, Antibiofilm, Antifungal, Antioxidant, and Wound-Healing Activities.

The purpose of this study is to create chitosan-stabilized silver nanoparticles (Chi/Ag-NPs) and determine whether they were cytotoxic and also to determine their characteristic antibacterial, antibiofilm, and wound healing activities. Recently, the development of an efficient and environmentally friendly method for synthesizing metal nanoparticles based on polysaccharides has attracted a lot of interest in the field of nanotechnology. Colloidal Chi/Ag-NPs are prepared by chemical reduction of silver ions in the presence of Chi, giving Chi/Ag-NPs. Physiochemical properties are determined by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) analyses. TEM pictures indicate that the generated Chi/Ag-NPs are nearly spherical in shape with a thin chitosan covering around the Ag core and had sizes in the range of 9–65 nm. In vitro antibacterial activity was evaluated against Staphylococcus aureus and Pseudomonas aeruginosa by a resazurin-mediated microtiter plate assay. The highest activity was observed with the lowest concentration of Chi/Ag-NPs, which was 12.5 µg/mL for both bacterial strains. Additionally, Chi/Ag-NPs showed promising antifungal features against Candida albicans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger, where inhibition zones were 22, 29, 20, and 17 mm, respectively. Likewise, Chi/Ag-NPs revealed potential antioxidant activity is 92, 90, and 75% at concentrations of 4000, 2000, and 1000 µg/mL, where the IC50 of Chi/Ag-NPs was 261 µg/mL. Wound healing results illustrated that fibroblasts advanced toward the opening to close the scratch wound by roughly 50.5% after a 24-h exposure to Chi/Ag-NPs, greatly accelerating the wound healing process. In conclusion, a nanocomposite based on AgNPs and chitosan was successfully prepared and exhibited antibacterial, antibiofilm, antifungal, antioxidant, and wound healing activities that can be used in the medical field.

1309-P: Central Mechanisms of Pollution-Induced Metabolic Disease

There is growing evidence linking exposure to volatile organic compounds (VOC) in ambient air with metabolic disease in humans. Benzene is a prominent VOC present in water, food, detergents, vehicle exhaust, tobacco smoke, and e-cigarettes. However, a direct link between exposure to VOC and metabolic homeostasis is not yet established. We performed a meta-analysis and found a positive association between exposure to VOC and metabolic impairments. Using a highly controlled mouse exposure system, we have recently demonstrated that chronic exposure of mice to VOC induces neuroinflammation and insulin resistance predisposing to T2D. To establish a timeline for the onset of VOC-triggered metabolic effects, we monitored glucose changes of exposed mice using continuous glucose monitoring (CGM) system. Surprisingly, we detected significant changes in glycemic control of male mice starting at day 4 of exposure. Further, mice exhibited impaired parameters of energy homeostasis within 6h of exposure. This was associated with activation of hypothalamic gliosis via stimulation of the NF-κB signaling and dysregulation of hypothalamic insulin signaling, contributing to blunted responses to glucose stimulation in glucose-sensing neurons. To determine the role of the central inflammatory mechanism in VOC-induced metabolic imbalance, we genetically ablated NF-κB signaling in microglia using Cx3cr1creERΔIKK mice. Selective inhibition of microglial NF-κB signaling completely restored hypothalamic insulin signaling, astrocytes activation, and hypothalamic glucose-sensing, consequently protecting from whole-body metabolic imbalance induced by VOC exposure. Importantly, this effect was mediated via hypothalamic ER stress response. Collectively, our data provide evidence that neuroinflammatory responses precede and mechanistically contribute to whole-body metabolic imbalance and identify air pollution-induced cellular and molecular targets in the hypothalamus that lead to metabolic disease. Disclosure L.K.Debarba: n/a. L.Koshko: None. H.M.Jayarathne: None. Z.Yi: None. U.Klueh: Research Support; Dexcom, Inc. M.Sadagurski: None. Funding American Diabetes Association (1-18-JDF-063)

Persulfate-based visible photocatalysis with a novel stability enhanced Fe-based metal-organic framework

Enhancing the water stability is the critical issue for metal–organic frameworks to explore their potential as photocatalysts for water treatment. In this study, a postsynthetic modification (PSM) method was applied, and MIL-101(Fe)–NH2 was modified to form a new MIL-101(Fe)-1-(4-(methyl)phenyl)urea (named MIL-101(Fe)-MPU). Chemical structure analyses confirmed a conversion from amino group to urea group. After PSM, grafting of a carbamido-benzene-methyl arm increased the surface hydrophobicity of MIL-101(Fe)-MPU with a large contact angle (> 100°). As a consequence, MIL-101(Fe)-MPU presented a better water stability compared to MIL-101(Fe)–NH2. After a 72-h liquid water exposure, chemical skeleton structure of MIL-101(Fe)-MPU were retained. In the presences of visible light and persulfate, both MIL-101(Fe)–NH2 and MIL-101(Fe)-MPU demonstrated equivalent photocatalysis degradation efficiencies of ciprofloxacin (CIP) in water matrix. The removal of CIP fit a quasi-first-order kinetic with apparent rate constants at 0.0440 min−1 (MIL-101(Fe)–NH2) and 0.0396 min−1 (MIL-101(Fe)-MPU). The dominant reaction mechanism in light/MIL-101(Fe)-MPU/S2O82− system was verified to be visible photocatalysis, including OH· and SO4·- oxidation. In general, MIL-101(Fe)-MPU presented an enhanced water stability, contributing to its potential photocatalysis applications in water treatments.

Molting enhances internal concentrations of fipronil and thereby decreases survival of two estuarine resident marine crustaceans.

In aquatic arthropods, molting is a pivotal physiological process for normal development, but it may also expose them to higher risks from xenobiotics, because the organism may take up additional water during that time. This study aimed to assess the effects of molting on bioconcentration and survival after 96-h exposure to insecticide fipronil with or without oxygenase (CYP450s) inhibitor piperonyl butoxide (PBO) of two estuarine resident marine crustacean species: the sand shrimp Crangon uritai and the kuruma prawn Penaeus japonicus, with 96-h LC50 value of fipronil=2.0µg/L and 0.2µg/L, respectively. Two graded concentrations included group high (H) (equivalent to the 96-h LC50 values) and low (L) (one-tenth of the H group concentration). Molting and survival were individually checked, and internal concentrations of fipronil and its metabolites (fipronil desulfinyl, fipronil sulfide, fipronil sulfone) were measured. The results showed that, only fipronil and fipronil sulfone were detected from organism, and that internal concentrations of these insecticides in molted specimens were higher than those of unmolted ones but comparable with those of dead ones. Accordingly, mortality was more frequent in molted specimens than those that were unmolted. Furthermore, involvement of oxygenase and higher lethal body burden threshold may confer higher tolerance to fipronil in sand shrimp than in the kuruma prawn. This study is the first to demonstrate that the body-residue-based approach is useful for deciphering the causal factors underlying fipronil toxicity, but highlights the need to consider physiological factors in arthropods, which influence and lie beyond body burden, molting and drug metabolism.

High-frequency ultrasound exposure improves depressive-like behavior in an olfactory bulbectomized rat model of depression.

ObjectivesAccording to previous studies, ultrasound exposure appears to be a noninvasive method for modulating brain activity related to cognition and consciousness; however, its effects on emotional states remain unclear. Therefore, an animal model is required in which the effects and effect mechanisms of ultrasound exposure can be investigated. Thus, we used olfactory bulbectomized rats as an animal model of depression and investigated their emotional state following ultrasound exposure.MethodsIn male Wistar/ST olfactory bulbectomized rats, hyperemotionality was evaluated according to hyperemotionality scoring and the scores before and after 24-h ultrasound exposure were compared. Elevated plus maze (EPM) tests were also conducted after 24-h ultrasound exposure, and blood samples were collected in which plasma corticosterone concentrations were measured.ResultsFollowing exposure to high-frequency (~50 kHz) ultrasound vocalizations (USVs) associated with the pleasant emotions of rats, the hyperemotionality scores of olfactory bulbectomized rats were significantly reduced. Additionally, the latency of the first entry into the open arm of the EPM was significantly decreased in USV-exposed olfactory bulbectomized rats, as were their plasma corticosterone levels. Furthermore, artificial ultrasound (50 kHz) at a similar frequency to that of USV also significantly decreased the hyperemotionality score of olfactory bulbectomized rats.ConclusionsUltrasound exposure improved depressive-like behavior in olfactory bulbectomized rats and reduced their plasma corticosterone levels. Thus, we recommend the use of olfactory bulbectomized rats as an animal model for investigating the effects and effect mechanisms of ultrasound exposure.

Oxidative stress responses in two marine diatoms during acute n-butyl acrylate exposure and the toxicological evaluation with the IBRv2 index

n-Butyl acrylate (nBA), a typical hazardous and noxious substance (HNS), is the largest-volume acrylate ester used to produce various types of polymers. With the increasing volume of nBA subject to maritime transportation, its accidental leakage poses a great risk to the marine organisms. Therefore, it is necessary to evaluate the ecological risk of nBA in marine environments. In this study, two species of marine microalgae, Skeletonema costatum and Phaeodactylum tricornutum, were used to explore the toxic effects of nBA based on their growth, pigment content, and oxidative stress. The growth of each species was significantly inhibited by nBA, showing a 96 h-EC50 value of 2.23 mg/L for P. tricornutum and 8.19 mg/L for S. costatum, respectively. Although chlorophylls a and c exerted a hormesis effect in P. tricornutum, contents of pigments generally decreased at high concentrations. In P. tricornutum, all detected antioxidants (reduced glutathione, GSH; superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx) were stimulated at concentrations ranging from 1.50 to 3.82 mg/L. However, these elevations were not enough to reduce the oxidative damage caused by nBA, because the content of malondialdehyde (MDA) increased continuously during 96-h exposure. For S. costatum, the activities of only two antioxidants (GSH and CAT) were enhanced, which is enough to prevent the MDA content from rising, even at higher concentrations of nBA (5–10 mg/L). The Integrated Biomarker Response Version 2 (IBRv2) index that combines responses of the above five oxidative stress biomarkers, was not only correlated positively with nBA concentration but could also indicate the occurrence of oxidative stress caused by acute concentration of nBA. These findings showed that P. tricornutum was sensitive to nBA compared to S. costatum, and the IBRv2 index was an effective tool for evaluating ecotoxicological effects on marine microalgae due to nBA spills.

Toxicity of Wildland Fire-Fighting Chemicals in Pulsed Exposures to Rainbow Trout and Fathead Minnows.

Intrusions of fire-fighting chemicals in streams can result from containment and suppression of wildfires and may be harmful to native biota. We investigated the toxicity of seven current-use fire-fighting chemicals to juvenile rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas) by simulating chemical intrusions under variable field conditions to provide insights into the potential damage these chemicals may cause in waterways. In three separate attenuated exsposure assays in which chemical concentration decreased throughout the 96-h exposure period, we variedwater flow rate, water hardness, and initial concentration of test chemical. In an additional series of four pulsed exposure assays in which fish encounter chemical for up to 1 h followed by an observation period in control water, we altered concentration of test chemical, water temperature, duration of chemical exposure, and number of exposures to determine delayed toxicity or recovery. Mortality of rainbow trout was higher across treatments at a warmer temperature and also increased with increasing concentration rate, increasing exposure duration, and sequential exposures across assays. For fathead minnows, mortality increased with increasing concentration of fire retardant and longer exposure durations. Because the ratio of toxic un-ionized ammonia to ionized ammonia is greater with increasing temperature and pH, future studies could investigate the effects of water temperature and pH on native fishes under environmentally relevant concentrations of fire-fighting chemicals. Environ Toxicol Chem 2022;41:1711-1720. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

PM2.5-Induced Programmed Myocardial Cell Death via mPTP Opening Results in Deteriorated Cardiac Function in HFpEF Mice.

PM2.5 exposure can induce or exacerbate heart failure and is associated with an increased risk of heart failure hospitalization and mortality; however, the underlying mechanisms remain unclear. This study focuses on the potential mechanisms underlying PM2.5 induction of cardiomyocyte programmed necrosis as well as its promotion of cardiac function impairment in a mouse model of heart failure with preserved ejection fraction (HFpEF). HFpEF mice were exposed to concentrated ambient PM2.5 (CAP) (CAP group) or filtered air (FA) (FA group) for 6weeks. Changes in myocardial pathology and cardiac function were documented for comparisons between the two groups. In vitro experiments were performed to measure oxidative stress and mitochondrial permeability transition pore (mPTP) dynamics in H9C2 cells following 24h exposure to PM2.5. Additionally, co-immunoprecipitation was conducted to detect p53 and cyclophilin D (CypD) interactions. The results showed exposure to CAP promoted cardiac function impairment in HFpEF mice. Myocardial pathology analysis and in vitro experiments demonstrated that PM2.5 led to mitochondrial damage in cardiomyocytes and, eventually, their necrosis. Moreover, our experiments also suggested that PM2.5 increases mitochondrial reactive oxygen species (ROS), induces DNA oxidative damage, and decreases the inner mitochondrial membrane potential (ΔΨm). This indicates the presence of mPTP opening. Co-immunoprecipitation results showed a p53/CypD interaction in the myocardial tissue of HFpEF mice in the CAP group. Inhibition of CypD by cyclosporin A was found to reverse PM2.5-induced mPTP opening and H9C2 cell death. In conclusion, PM2.5 induces mPTP opening to stimulate mitochondria-mediated programmed necrosis of cardiomyocytes, and it might exacerbate cardiac function impairment in HFpEF mice.

Abstract B030: Mimicking the evolution of doxorubicin resistance in triple negative breast cancer

Abstract Chemotherapy resistance presents a major challenge in the treatment of triple negative breast cancer (TNBC), which is characterized by low or no expression of the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor-2 (HER2). Because TNBC tumors do not express these receptors, there has been little to no advancement in the establishment of drug therapies to specifically target this breast cancer subtype. Additionally, unfavorable side effects and drug resistance reveal an urgent need for safer, more effective therapeutic options. In this study, we aim to mimic cancer recurrence by establishing doxorubicin (DOX) resistance in TNBC cell lines, HCC1806 and HCC70 and investigate the effect on cell survival, cell migration, and changes in gene expression. Here we show that TNBC cell growth was sustained after pulse treatments with increasing concentrations of DOX. HCC1806 and HCC70 cells treated with DOX titrants (0.5 µM-2.0 µM) demonstrate cell growth sustainability after 3h treatments. The DOX-treated TNBC cells also demonstrate anti-migratory potential. RNA sequencing analysis show that IL6, TP53, and TNFSF12 are uniquely expressed after 24h exposure to 0.5uM DOX. We plan to further investigate the roles these genes play in chemoresistance. Understanding changes in the gene network, as a result of doxorubicin-resistance, may help to establish biomarkers that could represent targets for drug therapies to overcome chemoresistance in TNBC. Citation Format: Kashenya Pearson, Terrence Everett, Checo J. Rorie. Mimicking the evolution of doxorubicin resistance in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B030.

Evaluation of oxidative stress and genotoxicity of 900MHz electromagnetic radiations using Trigonella foenum-graecum test system.

Unprecedented growth in the communication sector and expanded usage of the number of wireless devices in the past few decades have resulted in a tremendous increase in emissions of non-ionizing electromagnetic radiations (EMRs) in the environment. The widespread EMRs have induced many significant changes in biological systems leading to oxidative stress as well as DNA damage. Considering this, the present study was planned to study the effects of EMRs at 900MHz frequency with the power density of 10.0 dBm (0.01W) at variable exposure periods (0.5h, 1h, 2h, 4h, and 8h per day for 7days) on percentage germination, morphological characteristics, protein content, lipid peroxidation in terms of malondialdehyde content (MDA), and antioxidant defense system of Trigonella foenum-graecum test system. The genotoxicity was also evaluated using similar conditions. It was observed that EMRs significantly decreased the germination percentage at an exposure time of 4h and 8h. Fresh weight and dry weight of root and shoot did not show significant variations, while the root and shoot length have shown significant variations for 4h and 8h exposure period. Further, EMRs enhanced MDA indicating lipid peroxidation. In response to exposure of EMRs, there was a significant up-regulation in the activities of enzymes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione-S-transferase (GST), guaiacol peroxidase (POD), and glutathione reductase (GR) in the roots and shoots of Trigonella-foenum graecum. The genotoxicity study showed the induction of chromosomal aberrations in root tip cells of the Trigonella foenum-graecum test system. The present study revealed the induction of oxidative stress and genotoxicity of EMRs exposure in the test system.

The influence of methodology on the comparison of cytotoxicity of total-etch and self-etch adhesive systems

ObjectivesThe present study aimed to compare the in vitro cytocompatibility of two etch-and-rinse (Adper Scothbond, Optibond) and two self-etch (Clearfill SE Bond and Single Bond Universal) dental adhesives through a dentin-barrier model with human pulp fibroblasts. MethodsHuman fibroblasts were placed on a plastic device containing 500μm human dentin discs treated with each adhesive or without treatment (control). Other groups were directly exposed to media conditioned with adhesive samples according to ISO 10993-5:2009. After 24h exposure, cell viability was assessed by XTT, and released inflammatory mediators were detected with a multiparametric immunoassay. ResultsThe standardized test without barrier indicated both etch-and-rinse adhesives and self-etch as cytotoxic, promoting viabilities under 70% of the control group (p<0.05). The dentin-barrier model identified increased cell viability for self-etch adhesives, with Clearfill SE Bond identified as non-cytotoxic. The immunoassay evidenced high rates of cytokines by cells exposed to the conditioned media of Adper Scotchbond, Optibond S, and Single Bond Universal. ConclusionsThe use of a dentin-barrier in vitro model detected a better biocompatibility for self-etching adhesives and, in the case of Clearfill SE Bond, with a reversion from cytotoxic to biocompatible when compared to the indirect standardized test. Clinical significanceThe use of a dentin-barrier in vitro model was able to detect a better biocompatibility for self-etching adhesives when compared to the indirect standardized test and presents itself as a predictive in vitro method for assessing the cytotoxicity of dental restorative materials that may simulate the clinical condition more accurately.

Effects of Two Toxin-Producing Harmful Algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on Activity and Mortality of Larval Shellfish.

Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10–1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in >50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters.

Rapid and efficient testing of the toxicity of graphene-related materials in primary human lung cells

Graphene and its derivative materials are manufactured by numerous companies and research laboratories, during which processes they can come into contact with their handlers' physiological barriers—for instance, their respiratory system. Despite their potential toxicity, these materials have even been used in face masks to prevent COVID-19 transmission. The increasingly widespread use of these materials requires the design and implementation of appropriate, versatile, and accurate toxicological screening methods to guarantee their safety. Murine models are adequate, though limited when exploring different doses and lengths of exposure—as this increases the number of animals required, contrary to the Three R's principle in animal experimentation. This article proposes an in vitro model using primary, non-transformed normal human bronchial epithelial (NHBE) cells as an alternative to the most widely used model to date, the human lung tumor cell line A549. The model has been tested with three graphene derivatives—graphene oxide (GO), few-layer graphene (FLG), and small FLG (sFLG). We observed a cytotoxic effect (necrosis and apoptosis) at early (6- and 24-h) exposures, which intensified after seven days of contact between cells and the graphene-related materials (GRMs)—with cell death reaching 90% after a 5 µg/mL dose. A549 cells are more resistant to necrosis and apoptosis, yielding values less than half of NHBE cells at low concentrations of GRMs (between 0.05 and 5 µg/mL). Indeed, GRM-induced cell death in NHBE cells is comparable to that induced by toxic compounds such as diesel exhaust particles on the same cell line. We propose NHBE as a suitable model to test GRM-induced toxicity, allowing refinement of the dose concentrations and exposure timings for better-designed in vivo mouse assays.

A novel approach combining grain boundary engineering and grain boundary serration to enhance high-temperature hot corrosion resistance in Alloy 617

In this work, a novel idea of combining two established microstructural engineering approaches viz., grain boundary engineering (GBE) and grain boundary serration (GBS) through optimization of thermomechanical and thermal processing in Alloy 617 is investigated and the superior resistance of GBE+GBS microstructure to the high-temperature hot corrosion is demonstrated. To achieve the GBE+GBS microstructure, the GBS treatment was introduced as a part of the GBE processing schedule (i.e., incomplete GBE+GBS) and following the GBE treatment (i.e., complete GBE+GBS). The extent of GBS is found to be similar in all the processed specimens. However, the extent of GBE is observed to be lower in the specimens undergoing incomplete GBE+GBS. This is due to the occurrence of recrystallization and consequent infrequent multiple twinning. On the other hand, a higher extent of GBE is achieved in the specimens subjected to complete GBE+GBS owing to the retention of the optimized GBE microstructure following the GBS treatment. The synergistic influence of GBE and GBS on the hot corrosion behavior is assessed by exposing the as-received (AR) as well as optimized grain boundary engineered and serrated (GBES) specimens to a salt mixture at 1273 K. The percolation depth after 24h and 48h exposure is significantly lower in the GBES specimen (∼55 µm and ∼115 µm, respectively) than the AR condition (∼305 µm and ∼630 µm, respectively). This is ascribed to the incorporation of Σ3n (n ≤3) and serrated boundaries in the GBES microstructure which obstructed the infiltration of harmful species into the alloy.

Cu and Pb accumulation and removal from aqueous medium by Enydra fluctuans Lour. (Asteraceae) - a medicinal plant with potential for phytoremediation.

Enydra fluctuans Lour. (Asteraceae) is an edible semi-aquatic floating or trailing herbaceous plant widely distributed in tropical Africa, South and South East Asia, and Australia. Its leaves, which are consumed as a vegetable, are also used in traditional medicine to treat several diseases. The efficacy of this plant in removal of copper and lead from aqueous medium was tested in the present study. The plants were exposed to graded measured concentrations of 0.55-10.2mg Cu L-1 and 11.5-50.2mg Pb L-1 in hydroponic systems. Controls without added Cu and Pb were maintained under identical conditions. Cu and Pb were estimated by atomic absorption spectrometry, and the bioconcentration factor (BCF) and the translocation factor (TF) were calculated for each element at the different concentrations. Accumulation of both Cu and Pb was significantly higher in root than that in leaf and stem. Though all the bioconcentration factor (BCF) values were greater than unity, none of the translocation factor (TF) values was greater than unity, indicating that this plant could not be considered a hyperaccumulator of these metals. Nevertheless, E. fluctuans could remove Cu from aqueous medium at rates ranging from 98.8 to 99.7%, with a mean reduction of 99.2% after 96-h exposure at various concentrations. The removal of Pb ranged from 97.1 to 99.1%, with a mean reduction of 98.2%. Thus, E. fluctuans showed high potential for removal of Cu and Pb from aqueous medium and has the prospect of being used in phytoremediation of these metals.

Effects of Sodium Pyruvate on Vanadyl Sulphate-Induced Reactive Species Generation and Mitochondrial Destabilisation in CHO-K1 Cells.

Vanadium is ranked as one of the world’s critical metals considered important for economic growth with wide use in the steel industry. However, its production, applications, and emissions related to the combustion of vanadium-containing fuels are known to cause harm to the environment and human health. Pyruvate, i.e., a glucose metabolite, has been postulated as a compound with multiple cytoprotective properties, including antioxidant and anti-inflammatory effects. The aim of the present study was to examine the antioxidant potential of sodium pyruvate (4.5 mM) in vanadyl sulphate (VOSO4)-exposed CHO-K1 cells. Dichloro-dihydro-fluorescein diacetate and dihydrorhodamine 123 staining were performed to measure total and mitochondrial generation of reactive oxygen species (ROS), respectively. Furthermore, mitochondrial damage was investigated using MitoTell orange and JC-10 staining assays. We demonstrated that VOSO4 alone induced a significant rise in ROS starting from 1 h to 3 h after the treatment. Additionally, after 24 and 48 h of exposure, VOSO4 elicited both extensive hyperpolarisation and depolarisation of the mitochondrial membrane potential (MMP). The two-way ANOVA analysis of the results showed that, through antagonistic interaction, pyruvate prevented VOSO4-induced total ROS generation, which could be observed at the 3 h time point. In addition, through the independent action and antagonistic interaction with VOSO4, pyruvate provided a pronounced protective effect against VOSO4-mediated mitochondrial toxicity at 24-h exposure, i.e., prevention of VOSO4-induced hyperpolarisation and depolarisation of MMP. In conclusion, we found that pyruvate exerted cytoprotective effects against vanadium-induced toxicity at least in part by decreasing ROS generation and preserving mitochondrial functions

Non‐nutritive sweeteners competitively inhibit P‐glycoprotein in liver

Non‐nutritive sweeteners (NNS) are popular sugar replacements that provide sweet taste with little to no calories and are used in a wide range of products, including foods, beverages, and medications. Although NNS are recognized as safe for the general population at the Acceptable Daily Intake (ADI) by the Food and Drug Administration, the effects of NNS on specific patient populations and on human development are not completely understood. Previously, our lab has shown that in mice, pups exposed to NNS during early development present alterations in liver detoxification and metabolic enzymes. This led us to question whether NNS may disrupt detoxification in the liver, including transmembrane efflux transporters such as P‐glycoprotein (PGP). PGP plays a major role in the distribution of common xenobiotics and drugs, such as antibiotics, antidepressants, and antivirals, by excreting their conjugated metabolites from liver cells to the general circulation (blood and bile). Mutations in PGP, along with administration of pharmacological inhibitors lead to intracellular accumulation of its substrates and toxicity.We hypothesize that two common NNS, acesulfame potassium (AceK) and sucralose (Sucr), inhibit PGP’s efflux function in liver cells and alter PGP expression, suggesting reduced detoxification capacity in the liver.To investigate the effects of NNS on hepatic PGP, we first exposed the HepG2 liver carcinoma cell line to AceK and Sucr and assessed PGP mRNA expression by qPCR. Cells were treated with varying concentrations of AceK, Sucr, or combined NNS (AceK+Sucr) that spanned levels below and above the ADI for each sweetener for either 24h acute exposure or 72h chronic exposure. In parallel, PGP protein levels were assessed by Western blot. 24h combined exposure of AceK and Sucr, at concentrations above the ADI, increased PGP transcript expression, but had no effect at concentrations below or equal to the ADI. Only levels above the ADI of AceK and Sucr treatment at 72h led to increased PGP mRNA levels. Combined NNS treatment for 72h, however, increased PGP transcript levels at lower concentrations than acute exposure. In western blot analysis of protein expression, concentrations of combined NNS below the ADI increased PGP levels, in contrast to the qPCR data.As PGP inhibitor drugs have been shown to similarly affect its expression, we then wished to understand whether NNS impact PGP’s efflux action. Therefore, a cell‐free assay measuring PGP’s ATPase activity was used. Complementarily, in vitro assays of substrate retention and substrate efflux in HepG2 were performed to measure cellular efflux. We found that physiologically relevant NNS concentrations led to significant stimulation of ATPase activity, similar to potent PGP competitive inhibitors such as Verapamil. Similarly, low concentrations of AceK, Sucr, or combined NNS, comparable to 1/8th to 1 times the ADI for each, significantly retain PGP substrate Calcein‐AM intracellularly, or reduce efflux of PGP substrate Rhodamine 123, confirming PGP inhibition by NNS.Our results provide evidence that two common NNS, AceK and Sucr, are both substrates and inhibitors of PGP, and that both acute and chronic exposure to those NNS impacts PGP expression. Most importantly, altered PGP efflux function was observed after exposure of hepatic cells to sub‐ADI concentrations of NNS within expected exposure to common foods and beverages, suggesting risks for NNS consumers when taking medications that utilize the PGP as a main detoxification transporter.

Combined toxicity and toxicity persistence of antidepressants citalopram and mirtazapine to zooplankton Daphnia magna.

Citalopram (CTP) and mirtazapine (MTP) are two typical psychoactive drugs used for the depression treatment. As emerging pollutants, CTP and MTP have raised concern because of their harmful effect on aquatic organisms. Therefore, the ecotoxicological risk of these two pollutants to aquatic organisms should be given more attention. In this study, the effects of CTP and MTP on the feeding rate, heartbeat, nutritional enzymes, and their related gene expression of D. magna were investigated under single and binary mixture pollutant exposure. Subsequently, the recovery of exposed D. magna was studied to assess the toxic persistence of those pollutants. After 24-h exposure, the ingestion rate decreased by 34.2% and 21.5%, in the group of 1.45mg/L CTP (C-H) and binary mixture with high concentration (Mix-H), respectively. After 24-h recovery, the feeding rate of D. magna was stimulated by a compensatory response. Over the exposure period, the heartbeat rate of D. magna increased significantly in the groups of CTP, MTP, and their binary mixture with low concentration (Mix-L), and then, their heartbeat rate was recovered during the recovery period. The activity of α-amylase (AMS) and trypsin were significantly changed in most of the exposed daphnia, both during the exposure and recovery period. CTP/MTP exposure stimulated the expression of the AMS gene. MTP and Mix-H exposure inhibited the expression of the trypsin gene and the other groups stimulated its expression. After 24-h recovery, the stimulating or inhibitory effects were alleviated. There were different responses between gene expression and enzyme activity. In conclusion, our results highlighted the toxic effects at high concentrations of single and mixed pollution of CTP and MTP on the feeding rate, heartbeat, AMS and trypsin enzyme activity, and expression of related genes of D. magna to assess the environment risk of them.

Bioactivity of Deverra tortuosa essential oil, its nanoemulsion, and phenylpropanoids against the cowpea weevil, a stored grain pest with eco-toxicological evaluations.

The essential oil (EO) was hydrodistilled from of Deverra tortuosa aerial parts. Fifty-six components amounting 99.3% were identified in EO through using gas chromatography-flame ionization detection (GC-FID) and (GC-MS). Phenylpropanoids, dillapiole (41.6%), elemicin (7.3%) and myristicin (5.1%), and themonoterpene, sabinene (4.2%) were identified as the major terpenes. An oil-in-water nanoemulsion (particle size 70.3nm) was developed from EO adopting a low-energy method. The EO products showed insecticidal and biochemical effects against the cowpea weevil Callosobruchus maculatus. Based on a 48-h exposure period, the oil nanoemulsion exhibited a superior contact bioactivity (LC50 = 10.3µg/cm2), followed by EO (LC50 = 23.1µg/cm2), dillapiole (LC50 = 27.8µg/cm2), and myristicin (LC50 = 37.1µg/cm2). Upon fumigation, nanoemulsion and EO were superior as fumigants (LC50 after 48h were 6.9 and 14.3µl/l, respectively). Test materials showed a residual bioactivity against C. maculatus, where EO, dillapiole, and myristicin showed the strongest grain protecting activity. EO products significantly inhibited acetylcholinesterase (AChE) activity of C. maculatus adults. Test products were safe toward the non-target earthworms and did not alter the viability of cowpea seeds. There are evidences for the potential of using EO of D. tortuosa and its nanoemulsion and phenylpropanoids as natural grain protectants against C. maculatus.