Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

Exploring the Antimicrobial Potential and Biofilm Inhibitory Properties of Hemocyanin from Hemifusus pugilinus (Born, 1778).

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating ß-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1-5 µg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 µg/mL. Furthermore, H. pugilinus hemocyanin (10 µg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.

Adsorption Characteristics of Dimethylated Arsenicals on Iron Oxide-Modified Rice Husk Biochar.

In this study, the adsorption characteristics of dimethylated arsenicals to rice husk biochar (BC) and Fe/biochar composite (FeBC) were assessed through isothermal adsorption experiments and X-ray absorption spectroscopy analysis. The maximal adsorption capacities (qm) of inorganic arsenate, calculated using the Langmuir isotherm equation, were 1.28 and 6.32 mg/g for BC and FeBC, respectively. Moreover, dimethylated arsenicals did not adsorb to BC at all, and in the case of FeBC, qm values of dimethylarsinic acid (DMA(V)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) were calculated to be 7.08, 0.43, and 0.28 mg/g, respectively. This was due to the formation of iron oxide (i.e., two-line ferrihydrite) on the surface of BC. Linear combination fitting using As K-edge X-ray absorption near edge structure spectra confirmed that all chemical forms of dimethylated arsenicals adsorbed on the two-line ferrihydrite were DMA(V). Thus, FeBC could retain highly mobile and toxic arsenicals such as DMMTA(V) and DMDTA(V)) in the environment, and transform them into DMA(V) with relatively low toxicity.

Profiling Analysis of Filter Feeder Polypedilum (Chironomidae) Gut Contents Using eDNA Metabarcoding Following Contrasting Habitat Types-Weir and Stream.

We analyzed the dietary composition of Polypedilum larvae among two contrasting habitats (river and weir). Our approach was (i) to apply eDNA-based sampling to reveal the gut content of the chironomid larvae, (ii) the diversity of gut contents in the two aquatic habitats, and (iii) assessment of habitat sediment condition with the food sources in the gut. The most abundant food was Chlorophyta in the gut of the river (20%) and weir (39%) chironomids. The average ratio of fungi, protozoa, and zooplankton in river chironomids gut was 5.9%, 7.2%, and 3.8%, while it was found decreased to 1.2%, 2.5%, and 0.1% in weir chironomids. Aerobic fungi in river midge guts were 3.6% and 10.34% in SC and IS, while they were in the range of 0.34-2.58% in weir midges. The hierarchical clustering analysis showed a relationship of environmental factors with food contents. Abiotic factors (e.g., pH) in the river and weir habitats correlated the clustered pattern with phytoplankton and minor groups of fungi. This study could help understand the food source diversity in the chironomid and habitat environmental conditions by using eDNA metabarcoding as an effective tool to determine dietary composition.

Environmental co-exposure of high temperature and Cu induce hormonal disturbance of cortisol signaling and altered responses of cellular defense genes in zebrafish.

Global warming is causing a continuous increase in environmental temperatures, which simultaneously activates toxic environmental stresses, such as heavy metal exposure, in aquatic ecosystems. The present study aimed at evaluating the effects of Cu toxicity along with increased temperature during zebrafish embryogenesis. Decreased survival rates were observed following combined exposure to high temperature and Cu. Heart rates of zebrafish embryos were significantly increased only during heat stress. An abnormal morphology with curved body shape was induced by exposure to a combination of Cu and heat stress. Furthermore, heat stress also triggered Cu-induced intracellular reactive oxygen species (ROS) production, with upregulation of superoxide dismutase (SOD) and glutathione s-transferase (GST) expression, and cell death with modified expression of p53 and B-cell lymphoma-2 (Bcl-2) in zebrafish embryos. Finally, increased cortisol levels and altered expression of cortisol-signaling genes were observed following exposure to Cu and high temperatures. These results highlight that realistic exposure to combined stressors induces developmental disturbances via stress-induced responses involving oxidative stress and cell death as well as transcriptional alterations leading to cortisol signaling in fish.

A Small Molecule Promoting Neural Differentiation Suppresses Cancer Stem Cells in Colorectal Cancer.

Cancer stem cells (CSCs) are a tumor cell subpopulation that drives tumor progression and metastasis, leading to a poor overall survival of patients. In colorectal cancer (CRC), the hyper-activation of Wnt/ß-catenin signaling by a mutation of both adenomatous polyposis coli (APC) and K-Ras increases the size of the CSC population. We previously showed that CPD0857 inactivates Wnt/ß-catenin signaling by promoting the ubiquitin-dependent proteasomal degradation of ß-catenin and Ras proteins, thereby decreasing proliferation and increasing the apoptosis of CRC lines. CPD0857 also decreased the growth and invasiveness of CRC cells harboring mutant K-Ras resistant to EGFR mAb therapy. Here, we show that CPD0857 treatment decreases proliferation and increases the neuronal differentiation of neural progenitor cells (NPCs). CDP0857 effectively reduced the expression of CSC markers and suppressed self-renewal capacity. CPD0857 treatment also inhibited the proliferation and expression of CSC markers in D-K-Ras MT cells carrying K-Ras, APC and PI3K mutations, indicating the inhibition of PI3K/AKT signaling. Moreover, CPD0857-treated xenograft mice showed a regression of tumor growth and decreased numbers of CSCs in tumors. We conclude that CPD0857 could serve as the basis of a drug development strategy targeting CSCs activated through Wnt/ß-catenin-Ras MAPK-PI3K/AKT signaling in CRCs.

Cadmium-induced developmental alteration and upregulation of serine-type endopeptidase transcripts in wild freshwater populations of Chironomus plumosus.

Cadmium, a toxic heavy metal, is a persistent environmental contaminant with irreversible toxicity to aquatic organisms. Chironomus plumosus, a natural species, is the largest sediment-burrowing aquatic midge in freshwater environments. In this study, we evaluated developmental defects in C. plumosus resulting from Cd exposure. In C. plumosus larvae, Cd exposure induced decreased survival and growth rates, reduction of emergence rate and sex ratio, and delayed emergence, as well as elevating the incidence of split tooth deformities. To identify potential biomarker genes to assess environmental pollutants such as Cd, we identified differentially expressed genes (DEGs) in C. plumosus exposed to various Cd concentrations. Among fourteen characterized DEGs, serine-type endopeptidase (SP) and heat shock protein 70 (HSP70) genes exhibited significant upregulation in C. plumosus larvae after Cd exposure. Therefore, we evaluated SP and HSP70 responses in natural C. plumosus populations collected from three sites of a Korean river and analyzed their correlations with eighteen environmental quality characteristics using principal component analysis. The highest expression of SP and HSP70 transcripts was observed in C. plumosus populations from Yeosu in Korea, which has high concentrations of polluting heavy metals. SP transcript expression was positively correlated with concentrations of Cd, Pb, Al, Fe, NO2, and NO3. These results suggested that environmental pollutants such as Cd can impair proteolytic activity in the digestive system of C. plumosus and may ultimately induce developmental alterations. We therefore suggest SP as a potential biomarker to assess the effects of environmental pollutants in aquatic ecosystems.

Effects of combined stressors to cadmium and high temperature on antioxidant defense, apoptotic cell death, and DNA methylation in zebrafish (Danio rerio) embryos.

Fish are frequently affected by environmental stressors, such as temperature changes and heavy metal exposure, in aquatic ecosystems. In this study, we evaluated the combined effects of cadmium (Cd) toxicity and temperature (rearing temperature of 26 °C and heat stress at 34 °C) on zebrafish (Danio rerio) embryos. The survival and heart rates of zebrafish embryos decreased at relatively high Cd concentrations of 0.07 and 0.1 mg L-1. Abnormal morphology was induced by exposure to a combination of Cd toxicity and heat stress. The yolk sac edema size was not significantly different between the control- and Cd-treated groups. Cd exposure induced reactive oxygen species (ROS) production and cell death in the live zebrafish. High temperature (34 °C) triggered Cd-induced cell death and intracellular ROS production to a greater extent than the rearing temperature of 26 °C. Transcriptional levels of six genes-CAT, SOD, p53, BAX, Dnmt1, and Dnmt3b-were investigated. The mRNA expression of CAT and SOD, molecular indicators of oxidative stress, was increased significantly at 34 °C after Cd exposure. The mRNA expression of CAT was more sensitive to temperature than that of SOD in Cd-treated zebrafish. p53 and BAX, apoptosis-related genes, were upregulated upon combined exposure to high temperature and Cd. In addition, at 34 °C, the expression of Dnmt1 and Dnmt3b transcripts, markers of DNA methylation, was increased upon exposure of zebrafish to all concentrations of Cd. Overall, these results suggest that high temperature facilitates the potential role of Cd toxicity in the transcriptional regulation of genes involved in the antioxidant system, apoptosis, and DNA methylation.

An Aqueous Extract of Octopus ocellatus Meat Protects Hepatocytes Against H2O2-Induced Oxidative Stress via the Regulation of Bcl-2/Bax Signaling.

Octopus ocellatus meat (OM) is well known as a plentiful protein source. In this study, we evaluated the hepatoprotective effect of an aqueous extract of OM (OMA) against H2O2-triggered oxidative stress in human hepatocytes. First of all, taurine rich OMA showed a good ORAC value and reducing power and it was similar with that of ascorbic acid, which is known as a strong antioxidant. Also, OMA significantly improved H2O2-decreased cell viability by reducing the generation of intracellular reactive oxygen species (ROS) in hepatocytes. Interestingly, the stimulation of H2O2-induced the formations of apoptotic bodies and sub-G1 DNA content, whereas they were inhibited by the treatment with OMA. Furthermore, OMA regulated the protein expression levels of apoptotic molecules, such as Bax and Bcl-2. Taken together, this study suggests that OMA, which contains an abundant amount of taurine, protects hepatocytes from H2O2-triggered oxidative stress and might be a functional food material with hepatoprotective effects.

Endocrine-disrupting chemicals impair the innate immune prophenoloxidase system in the intertidal mud crab, Macrophthalmus japonicus.

Endocrine-disrupting chemicals (EDCs), xenobiotics that interfere with endogenous hormone function, have been studied for their impacts in aquatic environments. However, there is limited information about the potentially hazardous impact of bisphenol A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) on the marine environment. The aim of this study was to investigate the effects of BPA and DEHP on the immune response of the intertidal mud crab, Macrophthalmus japonicus. In order to examine immunological responses involving the prophenoloxidase (proPO) system, mRNA transcript and activity levels of six immune-related genes, including lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP), proPO, phenoloxidase (PO), peroxinectin (PE), serine protease inhibitor (Serpin), and trypsin (Tryp), were assessed in M. japonicus hepatopancreas and gills exposed to BPA or DEHP. Expression of immune genes generally decreased in M. japonicus hepatopancreas and gills exposed to all concentrations of BPA by days 4 and 7. However, at day 1, expression of Serpin and Tryp genes was significantly increased in M. japonicus hepatopancreas and gills exposed to BPA. For DEHP exposure, all genes, with the exception of Serpin, were significantly downregulated in M. japonicus gills. In the hepatopancreas, gene expression of PO, proPO, and LGBP increased at day 1, and then decreased by day 7, while mRNA expression of Serpin and Tryp exhibited up-regulation over all exposure periods. In addition, PE gene expression was upregulated in hepatopancreas at day 7 in a dose-dependent manner. Taken together, these results indicated that the crab immune responses were perturbed by exposure to BPA, and, in particular, DEHP.

Disrupting effects of antibiotic sulfathiazole on developmental process during sensitive life-cycle stage of Chironomus riparius.

Antibiotics in the environment are a concern due to their potential to harm humans and interrupt ecosystems. Sulfathiazole (STZ), a sulfonamide antibiotic, is commonly used in aquaculture and is typically found in aquatic ecosystems. We evaluated the ecological risk of STZ by examining biological, molecular and biochemical response in Chironomus riparius. Samples were exposed to STZ for 12, 24 and 96 h, and effects of STZ were evaluated at the molecular level by analyzing changes in gene expression related to the endocrine system, cellular stress response and enzyme activity of genes on antioxidant and detoxification pathways. STZ exposure induced significant effects on survival, growth and sex ratio of emergent adults and mouthpart deformity in C. riparius. STZ caused concentration and time-dependent toxicity in most of the selected biomarkers. STZ exposure leads to significant heat-shock response of protein genes (HSP70, HSP40, HSP90 and HSP27) and to disruption by up-regulating selected genes, including the ecdysone receptor gene, estrogen-related receptors, ultraspiracle and E74 early ecdysone-responsive gene. Furthermore, STZ induced alteration of enzyme activities on antioxidant and detoxification responses (catalase, superoxide dismutase, glutathione peroxidase and peroxidase) in C. riparius. By inducing oxidative stress, antibiotic STZ disturbs the endocrine system and produces adverse effects in growth processes of invertebrates.

The effect of temperature gradients on endocrine signaling and antioxidant gene expression during Chironomus riparius development.

Temperature is one of the most important environmental factors affecting the biological processes of aquatic species. To investigate the potential effects of temperature on the developmental processes of aquatic invertebrates, we analyzed biological and molecular transcriptional responses during Chironomus riparius development, including five stages spanning from embryo to adult stages. We assessed the temperature change-induced reduction of survival rate, changes in biological development including the male:female ratio in emerged adults, the success rates of pupation and emergence, and the developmental timing of pupation and emergence. The increased temperature induced expression of endocrine signaling genes, such as the ecdysone receptor, ultraspiracle (ortholog of the RXR), and the estrogen-related receptor in the fourth-instar larval and pupal stages of C. riparius development. Altered temperature also affected the activity of antioxidant genes, including catalase, peroxidase, glutathione peroxidase, and superoxide dismutase during the fourth-instar larval to adult stages of C. riparius development, as a result of altered development. Increased temperature during the fourth-instar larval stage increased oxidative stress in pupae and adults. Responses of antioxidant genes to increased temperature occurred in a developmental stage-dependent manner. However, reduced temperature did not induce the expression of antioxidant genes in a developmental stage-dependent manner, although it did induce oxidative stress during C. riparius development. Increased temperature also caused greater toxicity of di-ethylhexyl phthalate (DEHP) in fourth-instar larvae. Our findings suggest that altered temperatures may disturb the invertebrate hormone system and developmental processes by inducing oxidative stress in aquatic environments.