The size-dependent effects of nanoplastics in mouse primary hepatocytes from cells to molecules.

Nanoplastics (NPs) are easily ingested by organisms and their major accumulation organ was determined to be liver. To date, the size-dependent cytotoxicity of NPs on mammalian hepatocytes remains unclear. This study utilized mouse primary hepatocytes and catalase (CAT) as specific receptors to investigate the toxicity of NPs from cells to molecules, focusing on size-dependent effects. Results showed that the larger the particle size of NP at low doses (≤ 50 mg/L), the most pronounced inhibitory effect on hepatocyte viability. 20 nm NPs significantly inhibit cell viability only at high doses (100 mg/L). Larger NP particles (500 nm and 1000 nm) resulted in a massive release of lactate dehydrogenase (LDH) from the cell (cell membrane damage). Reactive oxygen species (ROS), superoxide dismutase (SOD) and CAT tests suggest that NPs disturbed the cellular antioxidant system. 20 nm NPs show great strength in oxidizing lipids and disrupting mitochondrial function compared to NPs of other particle sizes. The degree of inhibition of CAT activity by different sized NPs was coherent at the cellular and molecular levels, and NP-500 had the most impact. This suggests that the structure and microenvironment of the polypeptide chain in the vicinity of the CAT active site is more susceptible to proximity and alteration by NP-500. In addition, the smaller NPs are capable of inducing relaxation of CAT backbone, disruption of H-bonding and reduction of α-helix content, whereas the larger NPs cause contraction of CAT backbone and increase in α-helix content. All NPs induce CAT fluorescence sensitization and make the chromophore microenvironment hydrophobic. This study provides new insights for NP risk assessment and applications.

Cytotoxicity of emerging halophenylacetamide disinfection byproducts in drinking water: Mechanism and prediction.

Halophenylacetamides (HPAcAms) have been identified as a new group of nitrogenous aromatic disinfection byproducts (DBPs) in drinking water, but the toxicity mechanisms associated with HPAcAms remain almost completely unknown. In this work, the cytotoxicity of HPAcAms in human hepatoma (HepG2) cells was evaluated, intracellular oxidative stress/damage levels were analyzed, their binding interactions with antioxidative enzyme were explored, and a quantitative structure-activity relationship (QSAR) model was established. Results indicated that the EC50 values of HPAcAms ranged from 2353 µM to 9780 µM, and the isomeric structure as well as the type and number of halogen substitutions could obviously induce the change in the cytotoxicity of HPAcAms. Upon exposure to 2-(3,4-dichlorophenyl)acetamide (3,4-DCPAcAm), various important biomarkers linked to oxidative stress and damage, such as reactive oxygen species, 8­hydroxy-2-deoxyguanosine, and cell apoptosis, exhibited a significant increase in a dose-dependent manner. Moreover, 3,4-DCPAcAm could directly bind with Cu/Zn-superoxide dismutase and induce the alterations in the structure and activity, and the formation of complexes was predominantly influenced by the van der Waals force and hydrogen bonding. The QSAR model supported that the nucleophilic reactivity as well as the molecular compactness might be highly important in their cytotoxicity mechanisms in HepG2 cells, and 2-(2,4-dibromophenyl)acetamide and 2-(3,4-dibromophenyl)acetamide deserved particular attention in future studies due to the relatively higher predicted cytotoxicity. This study provided the first comprehensive investigation on the cytotoxicity mechanisms of HPAcAm DBPs.

Regulation mechanisms of ferric ions release from iron-loaded transferrin protein caused by nano-sized polystyrene plastics-induced conformational and structural changes.

Currently, the binding of iron-binding protein transferrin (TF) with NPs and their interaction mechanisms have not been completely elucidated yet. Here, we probed the conformation-dependent release of Fe ions from TF induced by nano-sized polystyrene plastics (PS-NPs) using dialysis, ICP-MS, multi-spectroscopic techniques, and computational simulation. The results showed that the release of free Fe ions from TF was activated after PS-NPs binding, which displayed a clear dose-effect correlation. PS-NPs binding can induce the unfolding and loosening of polypeptide chain and backbone of TF. Alongside this we found that the TF secondary structure was destroyed, thereby causing TF protein misfolding and denaturation. In parallel, PS-NPs interacted with the chromophores, resulting in the occurrence of fluorescence sensitization effects and the disruption of the surrounding micro-environment of aromatic amino acids. Also, the binding of PS-NPs induced the formation of new aggregates in the PS-NPs-TF system. Further simulations indicated that PS-NPs exhibited a preference for binding to the hinge region that connects the C-lobe and N-lobe, which is responsible for the Fe ions release and structural alterations of TF. This finding provides a new understanding about the regulation of the release of Fe ions of iron-loaded TF through NPs-induced conformational and structural changes.

New mechanistic insights into halogen-dependent cytotoxic pattern of monohaloacetamide disinfection byproducts.

As highly toxic nitrogenous disinfection byproducts (DBPs), monohaloacetamides (monoHAcAms) generally exhibited a cytotoxic rank order of iodoacetamide ˃ bromoacetamide ˃ chloroacetamide. However, the mechanisms underlying the halogen-dependent cytotoxic pattern remain largely veiled as yet. In this work, oxidative stress/damage levels in monoHAcAm-treated Chinese hamster ovary cells were thoroughly analyzed, and binding interactions between monoHAcAms and antioxidative enzyme Cu/Zn-superoxide dismutase (Cu/Zn-SOD) were investigated by multiple spectroscopic techniques and molecular docking. Upon exposure to monoHAcAms, the intracellular levels of key biomarkers associated with oxidative stress/damage, including reactive oxygen species, malondialdehyde, lactate dehydrogenase, 8-hydroxy-2-deoxyguanosine, cell apoptosis, and G1 cell cycle arrest, were all significantly increased in a dose-response manner with the same halogen-dependent rank order as their cytotoxicity. Moreover, this rank order was also determined to be applicable to the monoHAcAm-induced alterations in the conformation, secondary structure, and activity of Cu/Zn-SOD, the microenvironment surrounding aromatic amino acid residues in Cu/Zn-SOD, as well as the predicted binding energy of SOD-monoHAcAm interactions. Our results revealed that the halogen-dependent cytotoxic pattern of monoHAcAms was attributed to their differential capacity to induce oxidative stress/damage and their interaction with antioxidative enzyme, which contribute to a better understanding of the halogenated DBP-induced toxicological mechanisms.

Novel evidence on iodoacetic acid-induced immune protein functional and conformational changes: Focusing on cellular and molecular aspects.

Elevated levels of iodide occur in raw water in certain regions, where iodination disinfection byproducts are formed during chloramine-assisted disinfection of naturally iodide-containing water. Iodoacetic acid (IAA) is one of the typical harmful products. The mechanisms underlying IAA-induced immunotoxicity and its direct effects on biomolecules remained unclear in the past. Cellular, biochemical, and molecular methods were used to investigate the mechanism of IAA-induced immunotoxicity and its binding to lysozyme. In the presence of IAA, the cell viability of coelomocytes was significantly reduced to 70.8 %, as was the intracellular lysozyme activity. Upon binding to IAA, lysozyme underwent structural and conformational changes, causing elongation and unfolding of the protein due to loosening of the backbone and polypeptide chains. IAA effectively quenched the fluorescence of lysozyme and induced a reduction in particle sizes. Molecular docking revealed that the catalytic residue, Glu 35, which is crucial for lysozyme activity, resided within the docking range, suggesting the preferential binding of IAA to the active site of lysozyme. Moreover, electrostatic interaction emerged as the primary driving force behind the interaction between IAA and lysozyme. In conclusion, the structural and conformational changes induced by IAA in lysozyme resulted in impaired immune protein function in coelomocytes, leading to cellular dysfunction.

Compound effect and mechanism of oxidative damage induced by nanoplastics and benzo [a] pyrene.

Nanoplastics and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soil environments. In order to objectively evaluate the toxic interaction between polystyrene nanoplastics (PS NPs) and benzo [a] pyrene (BaP), oxidative damage at the level of earthworm cells and biomacromolecules was investigated by experiments combined with molecular dynamics simulation. Studies on cells reveal that PS NPs and BaP had synergistic toxicity when it came to causing oxidative stress. Cellular reactive oxygen species (ROS) levels under combined pollutant exposure were 24% and 19% higher, respectively than when PS NPs and BaP were exposed alone (compared to the blank group). In addition, BaP and PS NPs inhibited the ability of CAT to decompose H2O2 by affecting the structure of the proximal amino acid Tyr 357 in the active center of CAT, which exacerbated oxidative stress to a certain extent. Therefore, the synergistic toxic effect of BaP and PS NPs is due to the mutual complement of the two to the induction of protein structural looseness, and the strengthening of the stability of the conjugate (CAT-BaP-PS) under the weak interaction. This work provides a new perspective and approach on how to talk about the toxicity of combined pollutants.

Decoding the biological toxicity of phenanthrene on intestinal cells of Eisenia fetida: Effects, toxicity pathways and corresponding mechanisms.

Phenanthrene is frequently detected and exists extensively in the soil environment, and its residues inevitably impose a significant threat to soil organisms. Exposure to and toxicity of phenanthrene on earthworms has been extensively studied before, however, the possible mechanisms and related pathways associated with phenanthrene-triggered toxicity at the intestinal cell level remain unclear. Herein, primary intestinal cells isolated from Eisenia fetida (Annelida, Oligochaeta) intestine were used as targeted receptors to probe the molecular mechanisms involved in ROS-mediated damaging effects and the potential pathways of phenanthrene-induced toxicity at cellular and sub-cellular levels. Results indicated that phenanthrene exposure induced oxidative stress by activating intracellular ROS (elevated O2-, H2O2, and OH- content) bursts in E. fetida intestinal cells, causing various oxidative damage effects, including lipid peroxidation (increased MDA content), protein oxidation (enhanced PCO levels), and DNA damage (enhanced 8-OHdG levels). The enzymatic and non-enzymatic strategies in earthworm cells were activated to mitigate these detrimental effects by regulating ROS-mediated pathways involving defense regulation. Also, phenanthrene stress destroyed the cell membrane of E. fetida intestinal cells, resulting in cellular calcium homeostasis disruption and cellular energetic alteration, ultimately causing cytotoxicity and cell apoptosis/death. More importantly, the mitochondrial dysfunction in E. fetida cells was induced by phenanthrene-caused mitochondrial membrane depolarization, which in turn caused un-controlled ROS burst and induced apoptosis through mitochondria-mediated caspase-3 activation and ROS-mediated mitochondrial-dependent pathway. Furthermore, exposure to phenanthrene activated an abnormal mRNA expression profile associated with defense regulation (e.g., Hsp70, MT, CRT, SOD, CAT, and GST genes) in E. fetida intestinal cells, resulting in various cellular dysfunctions and pathological conditions, eventually, apoptotic cell death. Taken together, this study offers valuable insights for probing the toxic effects and underlying mechanisms posed by phenanthrene at the intestinal cell level, and is of great significance to estimate the detrimental side effects of phenanthrene on soil ecological health.

Antiangiogenic potential of Lepista nuda extract suppressing MAPK/p38 signaling-mediated developmental angiogenesis in zebrafish and HUVECs.

The medicinal properties of natural/edible plant products and their use are popular in traditional practice owing to their nutritional contents with little to no side effects. Lepista nuda (L. nuda), an edible mushroom (Clitocybe nuda, commonly known as blewit), has attracted researchers to evaluate its contents and the mechanism of its activities. In the current study, we focused on evaluating the antiangiogenic effects of L. nuda water extract on zebrafish development and in vitro human umbilical vein endothelial cell (HUVEC) tube formation. Bioactive components such as ergothioneine, eritadenine, and adenosine were identified and quantified by HPLC analysis. The L. nuda extract showed antiangiogenic properties and inhibited intersegmental vessel (ISV), caudal vein plexus (CVP), hyaloid vessel (HV), and subintestinal vessel (SIV) development in Tg (fli1: EGFP) zebrafish embryos. The expression of angiogenesis-related genes (vegfaa, kdrl, vegfba, flt1, kdr) was affected following L. nuda extract treatment. L. nuda extract attenuated in vitro HUVEC tube formation, migration, and invasion. Furthermore, inhibition of MAPK/p38 signaling and depletion of proangiogenic genes, including growth factors (fgf, ang2, and vegfa); primary and accessory receptors (tie2, vegfr2, and eng); MMPs (mmp1 and mmp2); and cytokines (il-1α, il-1ß, il-6, and tnf-α) was observed in HUVECs following L. nuda treatment. An in vivo zebrafish xenograft assay showed that L. nuda extract inhibited HuCCT1 cell-induced SIV sprouting in HuCCT1-injected embryos. Collectively, the results suggest that L. nuda could be a potential inhibitor of angiogenesis limiting cancer progression.

The culprits of superoxide dismutase inactivation under size-dependent stress of ultrafine carbon black: Superoxide anion, genotoxicity and protein corona.

As a critical component of atmospheric ultrafine particulates, ultrafine carbon black (UFCB) brings great exposure risk to organisms. At present, the action pathway and activity regulation mechanism of UFCB on functional proteins in vivo are not clear, and the size-dependent effects of UFCB during this process need to be elucidated. Superoxide dismutase (SOD), one of the most applied biomarkers to assess the environmental impact of pollutants, plays crucial roles in resistance to oxidative stress. Here, based on the inactivation of SOD (84.79 %, 86.81 % and 91.70 %) in primary mouse hepatocytes exposed to UFCB (13 nm, 50 nm and 95 nm), oxidative stress, genotoxicity and protein molecular studies were employed to elucidate the inactivation mechanisms. Results showed that inhibition of UFCB-mediated superoxide anion (O2-) contributed to a decrease in SOD activity. Furthermore, the significant increase in 8-hydroxy-2-deoxyguanosine content and the comet tail formation indicated the occurrence of DNA damage, supporting that concomitant aberrant transcriptional and protein translational under gene regulation should be responsible for SOD inactivation. At the molecular level, the constricted backbone, reduced content of α-helix and fluorescence sensitization all demonstrated that the attachment-type binding of SOD on UFCB to form the 'protein corona' disrupted protein structure. Enzyme activity assays indicated that SOD backbone tightening and helix decay resulted in decreased activity, which should be another reason for intracellular SOD inactivation. More importantly, the particle sizes of UFCB exert powerful influences on SOD inactivation mechanisms. Smaller UFCB (13 nm) induced more severe O2- inhibition and DNA damage, while UFCB50nm with the best dispersity bound more SOD and induced stronger molecular toxicity, which are their different strengths in stressing SOD inactivation in hepatocytes. Our findings provide novel insights for exploring functional proteins activity and underscore a potentially size-dependent risk of nanoparticles.

Occurrence and Cytotoxicity of Aliphatic and Aromatic Halogenated Disinfection Byproducts in Indoor Swimming Pool Water and Their Incoming Tap Water.

Disinfection byproducts (DBPs) in swimming pool water are of wide concern for public health. In this study, the occurrence of five categories of aliphatic halogenated DBPs, i.e., trihalomethanes (THMs), haloacetic acids (HAAs), haloacetonitriles (HANs), halonitromethanes (HNMs), and haloketones (HKs), and six categories of aromatic halogenated DBPs, i.e., halophenols (HPs), halonitrophenols (HNPs), halohydroxy-benzaldehydes (HBALs), halohydroxybenzoic acids (HBAs), halobenzoquinones (HBQs), and haloanilines (HAs), was examined in seven indoor swimming pool water and their incoming tap water. The correlations between the DBP concentrations and water quality parameters were explored. Moreover, the cytotoxicity of the aliphatic and aromatic halogenated DBPs was tested with human hepatoma (HepG2) cells, and the concentration-cytotoxicity contributions of different DBP categories were calculated. The results demonstrate that 24 aliphatic (5 THMs, 8 HAAs, 5 HANs, 4 HNMs, and 2 HKs) and 50 aromatic halogenated DBPs (9 HPs, 8 HNPs, 9 HBALs, 8 HBAs, 11 HBQs, and 5 HAs) were present in the swimming pool water, among which 41 aromatic halogenated DBPs were detected in swimming pool water for the first time. The average concentrations of the five categories of aliphatic halogenated DBPs in the swimming pool water were in the order of HAAs > HANs > HKs > THMs > HNMs, while those in their incoming tap water were in the order of THMs > HAAs > HKs > HANs > HNMs. The average concentrations of the aromatic halogenated DBPs in the swimming pool water were significantly lower than those of the aliphatic halogenated DBPs, following the order of HBQs > HPs > HBAs > HBALs > HAs > HNPs, while those in their incoming tap water were in the order of HBALs > HBQs > HPs > HBAs > HAs > HNPs. The average concentration-cytotoxicity contributions of different DBP categories in the swimming pool water followed the order of HAAs > HANs > HNMs > HKs > HBQs > THMs > HPs > HNPs > HBAs > HBALs > HAs, with HAAs, HANs, and HNMs possessing the main concentration-cytotoxicity contributions (93.2% in total) among all DBP categories.

Research on Trajectory Recognition and Control Technology of Real-Time Tracking Welding.

Real-time tracking welding with the assistance of structured light vision enhances the intelligence of robotic welding, which significantly shortens teaching time and guarantees accuracy for user-customized product welding. However, the robustness of most image processing algorithms is deficient during welding practice, and the security regime for tracking welding is not considered in most trajectory recognition and control algorithms. For these two problems, an adaptive feature extraction algorithm was proposed, which can accurately extract the seam center from the continuous, discontinuous or fluctuating laser stripes identified and located by the CNN model, while the prior model can quickly remove a large amount of noise and interference except the stripes, greatly improving the extraction accuracy and processing speed of the algorithm. Additionally, the embedded Pauta criterion was used to segmentally process the center point data stream and to cyclically eliminate outliers and further ensure the accuracy of the welding reference point. Experimental results showed that under the guarantee of the above-mentioned seam center point extraction and correction algorithms, the tracking average error was 0.1 mm, and even if abnormal trajectory points existed, they did not cause welding torch shaking, system interruption or other accidents.

Occurrence and transformation of newly discovered 2-bromo-6-chloro-1,4-benzoquinone in chlorinated drinking water.

Halobenzoquinones (HBQs) have been reported as an emerging category of disinfection byproducts (DBPs) in drinking water with relatively high toxicity, and the previously reported HBQs include 2,6-dichloro-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2,6-diiodo-1,4-benzoquinone, 2-chloro-6-iodo-1,4-benzoquinone, and 2-bromo-6-iodo-1,4-benzoquinone. In this study, another HBQ species, 2-bromo-6-chloro-1,4-benzoquinone (2,6-BCBQ), was newly detected and identified in drinking water. The occurrence frequency and levels of 2,6-BCBQ were investigated, and its cytotoxicity was evaluated. Since the formed 2,6-BCBQ was found to be not stable in chlorination, its transformation kinetics and mechanisms in chlorination were further studied. The results reveal that 2,6-BCBQ was generated from Suwannee River humic acid with concentrations in the range of 4.4-47.9 ng/L during chlorination within 120 h, and it was present in all the tap water samples with concentrations ranging from 1.5 to 15.7 ng/L. Among all the tested bromochloro-DBPs, 2,6-BCBQ showed the highest cytotoxicity on the human hepatoma cells. The transformation of 2,6-BCBQ in chlorination followed a pseudo-first-order decay, which was significantly affected by the chlorine dose, pH, and temperature. Seven polar chlorinated and brominated intermediates (including HBQs, halohydroxybenzoquinones, and halohydroxycyclopentenediones) were detected in chlorinated 2,6-BCBQ samples, according to which the transformation pathways of 2,6-BCBQ in chlorination were proposed. Besides, four trihalomethanes and four haloacetic acids were also generated during chlorination of 2,6-BCBQ with molar transformation percentages of 1.6-13.7%.

Halohydroxybenzonitriles as a new group of halogenated aromatic DBPs in drinking water: Are they of comparable risk to halonitrophenols?

Halogenated aromatic disinfection byproducts (DBPs) exhibited similar total organic halogen levels in chlorinated drinking water samples as compared with aliphatic ones, and they predominantly accounted for the overall toxicity of the samples. Among the reported halogenated aromatic DBPs, halonitrophenols (HNPs) have received particular attention in recent years due to the relatively high risk in drinking water. In this study, a new group of halogenated aromatic DBPs were detected and then proposed to be halohydroxybenzonitriles (HHBNs) by employing the ultra-performance liquid chromatography/tandem mass spectrometers. Thereafter, the specific HHBN species in drinking water were theoretically speculated and then thoroughly identified with standard compounds. Their occurrence in drinking water was investigated, their cytotoxicity was evaluated, and their stability in the presence of chlorine was assessed. Seven newly identified HHBNs, including 3,5-dichloro-4-hydroxybenzonitrile, 3,5-dichloro-2-hydroxybenzonitrile, 5-bromo-3-chloro-4-hydroxybenzonitrile, 5-bromo-3-chloro-2-hydroxybenzonitrile, 3,5-dibromo-4-hydroxybenzonitrile, 3,5-dibromo-2-hydroxybenzonitrile, and 3,5-diiodo-4-hydroxybenzonitrile, showed 100% detection frequency in the collected drinking water samples with concentrations up to 36 ng/L. HHBNs exhibited significantly higher cytotoxicity in Chinese hamster ovary cells than regulated DBPs (e.g., trihalomethanes and haloacetic acids), which might be contributed by their cellular uptake efficiency and nucleophilicity. The seven HHBNs were proved to undergo transformation during chlorination following pseudo-first-order decay with half-lives in the range of 9-63 h. More importantly, in comparison to HNPs, which showed relatively high toxicity and strong stability among the halogenated aromatic DBPs, HHBNs presented comparable concentration-cytotoxicity contribution (50%) and slightly weaker stability (43%), suggesting that HHBNs should be a new group of DBPs of concern in drinking water.

Improvement in the probiotic efficacy of Bacillus subtilis E20-stimulates growth and health status of white shrimp, Litopenaeus vannamei via encapsulation in alginate and coated with chitosan.

The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5-2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg-1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg-1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg-1, shrimp's growth performance and health status improved after being fed 107 CFU kg-1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg-1) was able to increase the growth performance and health status of shrimp.

Formation and Cytotoxicity of Halophenylacetamides: A New Group of Nitrogenous Aromatic Halogenated Disinfection Byproducts in Drinking Water.

Nitrogenous aromatic halogenated disinfection byproducts (DBPs) in drinking water have received considerable attention recently owing to their relatively high toxicity. In this study, a new group of nitrogenous aromatic halogenated disinfection byproducts, halophenylacetamides (HPAcAms), were successfully identified for the first time in both the laboratory experiments and realistic drinking water. The formation mechanism of HPAcAms during chlorination of phenylalanine in the presence of Br- and I-, occurrence frequencies, and concentrations in authentic drinking water were investigated, and a quantitative structure-activity relationship (QSAR) model was developed based on the acquired cytotoxicity data. The results demonstrated that HPAcAms could be formed from phenylalanine in chlorination via electrophilic substitution, decarboxylation, hydrochloric acid elimination, and hydrolysis. The HPAcAm yields from phenylalanine were significantly affected by contact time, pH, chlorine dose, and temperature. Nine HPAcAms with concentrations in the range of 0.02-1.54 ng/L were detected in authentic drinking water samples. Most tested HPAcAms showed significantly higher cytotoxicity compared with dichloroacetamide, which is the most abundant aliphatic haloacetamide DBP. The QSAR model demonstrated that the cellular uptake efficiency and the polarized distributions of electrons of HPAcAms play essential roles in their cytotoxicity mechanisms.

Formation of chlorinated halobenzoquinones during chlorination of free aromatic amino acids.

Chlorinated halobenzoquinones (HBQs) widely exist in drinking water as emerging disinfection byproducts (DBPs), which have attracted significant attention due to their wide occurrence and high toxicity. In this study, the formation of chlorinated HBQs from the three free aromatic amino acids, tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe), during chlorination was investigated, the formation pathways of chlorinated HBQs were explained based on the detected intermediates and influence factors. The results revealed that four chlorinated HBQs, including 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,3,5-trichloro-1,4-benzoquinone, 2,3,5,6-tetrachloro-1,4-benzoquinone and 2,6-dichloro-3-methyl-1,4-benzonquinone, were formed in chlorination of the three free aromatic amino acids, and 2,6-DCBQ was the dominant species among the formed chlorinated HBQs. Of the three free aromatic amino acids, Trp and Tyr presented relatively high yields of chlorinated HBQs than Phe. Moreover, ten intermediates were successfully detected (e.g., N,2-dichloroaniline from Trp, 2,4,6-trichlorophenol from Tyr) according to the isotope and fragment information obtained using high resolution mass spectrometry. The formation pathways of chlorinated HBQs from Trp and Tyr were proposed to include electrophilic addition, electrophilic substitution, oxidation, deacidification and dehydration reaction, and further validated using theoretical calculation. The yields of chlorinated HBQs during chlorination of the free aromatic amino acids were significantly affected by free chlorine dosage, pH and temperature.

A synbiotic containing prebiotic prepared from a by-product of king oyster mushroom, Pleurotus eryngii and probiotic, Lactobacillus plantarum incorporated in diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei.

This study aimed to evaluate the effects of a synbiotic composite an extract from a by-product of king oyster mushroom, Pleurotus eryngii (KOME), and probiotic Lactobacillus plantarum 7-40 on the growth performance and health status of white shrimp, Litopenaeus vannamei. The KOME was able to stimulate the growth of probiotic, but not the growth of Vibrio pathogens, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. Four diets were formulated, including a control diet supplemented without prebiotic and probiotic, a basal diet supplemented with KOME (5 g kg-1) (ME), a basal diet supplemented with probiotic (1 × 108 CFU kg-1) (LP), and a basal diet supplemented with KOME (5 g kg-1) and probiotic (1 × 108 CFU kg-1) (SYN). Shrimp fed the ME, LP, and SYN diets had significantly higher survival than that of shrimp fed with the control diet for 8 weeks. Shrimp in the SYN group also had a significantly higher weight gain and total final weight in comparison with the control and other treatments. In the intestinal tract, lactic acid bacteria count was significantly higher in the SYN group, whereas the Vibrio-like bacteria count was significantly higher in the ME group than in the control group. For the health status assessment, the disease resistance of shrimp against V. alginolyticus was improved in all treatments compared to the shrimp in control. Shrimps in the SYN group had significantly lower cumulative mortality due to the significant increase in immune responses, including phenoloxidase, respiratory burst, and lysozyme activity, and the gene expression of pexn and pen4 in the haemocytes, and lgbp, sp, propoii, pexn, pen3a, pen4, and gpx in the haepatopancreas of shrimp as compared to the control. Therefore, it is suggested that a combination of KOME and probiotics can be used as a synbiotic to improve the growth performance and reduce the risk of infectious diseases caused by Vibrio and at the same time significantly contribute to the circular economy.

Haloquinone Chloroimides as Toxic Disinfection Byproducts Identified in Drinking Water.

Haloquinone chloroimides (HQCs) are suspected to be highly toxic contaminants, and their production during drinking water disinfection is predicted. However, HQC disinfection byproducts (DBPs) have not been reported in drinking water to date because of analytical limitations. In this study, we developed an analytical method to detect five HQCs, including 2,6-dichloroquinone-4-chloroimide (2,6-DCQC), 2,6-dibromoquinone-4-chloroimide (2,6-DBQC), 2-chloroquinone-4-chloroimide (2-CQC), 3-chloroquinone-4-chloroimide (3-CQC), and 2,6-dichloroquinone-3-methyl-chloroimide (2,6-DCMQC). This method combined a derivatization reaction of HQCs with phenol in alkaline solutions to produce halogenated indophenols, a solid-phase extraction pretreatment using hydrophilic-lipophilic balanced (HLB) cartridges, and a multiple reaction monitoring (MRM) method for quantification. The method was demonstrated to be sensitive and accurate with recoveries of 71-85% and limits of detection of 0.1-0.2 ng/L for the five tested HQCs. Using this method, five tested HQCs were identified in drinking water samples from nine water treatment plants and water distribution systems as new DBPs at concentrations of up to 23.1 ng/L. The cytotoxicity of the five tested HQCs in HepG2 cells was higher than or comparable to that of 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), an emerging DBP that was hundreds to thousands of times more toxic than regulated DBPs. This study presents the first analytical method for HQC DBPs in drinking water and the first set of occurrence and cytotoxicity data of HQC DBPs.

Probiotic Bacillus safensis NPUST1 Administration Improves Growth Performance, Gut Microbiota, and Innate Immunity against Streptococcus iniae in Nile tilapia (Oreochromis niloticus).

Probiotics are considered ecofriendly alternatives to antibiotics as immunostimulants against pathogen infections in aquaculture. In the present study, protease-, amylase-, cellulase-, and xylanase-producing Bacillus safensis NPUST1 were isolated from the gut of Nile tilapia, and the beneficial effects of B. safensis NPUST1 on growth, innate immunity, disease resistance and gut microbiota in Nile tilapia were evaluated by feeding tilapia a basal diet or basal diet containing 105 and 106-107 CFU/g for 8 weeks. The results showed that the weight gain, feed efficiency and specific growth rate were significantly increased in tilapia fed a diet containing 106 CFU/g and 107 CFU/g B. safensis NPUST1. Intestinal digestive enzymes, including protease, amylase and lipase, and hepatic mRNA expression of glucose metabolism and growth-related genes, such as GK, G6Pase, GHR and IGF-1, were also significantly increased in the 106 CFU/g and 107 CFU/g B. safensis NPUST1 treated groups. Immune parameters such as phagocytic activity, respiratory burst and superoxide dismutase activity in head kidney leukocytes, serum lysozyme, and the mRNA expression of IL-1ß, IL-8, TNF-α and lysozyme genes were significantly induced in the head kidney and spleen of 106 CFU/g and 107 CFU/g B. safensis NPUST1 treated fish. The cumulative survival rate was significantly increased in fish fed a diet containing 106 CFU/g and 107 CFU/g B. safensis NPUST1 after challenge with Streptococcus iniae. Dietary supplementation with B. safensis NPUST1 improves the gut microbiota of Nile tilapia, which increases the abundance of potential probiotics and reduces the abundance of pathogenic pathogens. The present study is the first to report the use of B. safensis as a potential probiotic in aquaculture, and a diet containing 106 CFU/g B. safensis NPUST1 is adequate for providing beneficial effects on growth performance and health status in tilapia.

Effect of Tempeh on Gut Microbiota and Anti-Stress Activity in Zebrafish.

Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg-1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light-dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacteriumadolescentis, a "psychobiotic", increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.