Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels.

In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.

Negative nanopore sequencing for mapping biochemical processes on DNA molecules.

Nanopore sequencing maps biochemical processes on DNA by detecting negative peaks in the sequence alignment profile. Protein-bound DNA and single-strand broken DNA cannot pass through nanopores, resulting in unaligned regions in the genome MAP. This novel approach provides a clear representation of genomic biochemical events.

Large contributions of petrogenic and aged soil-derived organic carbon to Arctic fjord sediments in Svalbard.

Svalbard fjords are recognized as hotspots for organic carbon (OC) burial and storage due to their high sedimentation rates, which effectively trap terrestrial sediments and inhibit extensive OC remineralization. In this study, we investigated surface sediments (n = 48) from eight Svalbard fjords, along with bedrock (n = 17), soil (n = 28), and plant (n = 12) samples, to identify the sources of sedimentary OC in these fjords using geochemical parameters. All examined surface sediments from the fjords showed a depletion in 14Corg (- 666.9 ± 240.3‰), indicating that recently fixed terrestrial and marine biomass alone cannot account for the entire sedimentary OC pool. Conventional bulk indicators such as Norg/TOC ratio and δ13Corg were insufficient for fully determining the sources of sedimentary OC. Therefore, we employed a four-end-member approach, using Δ14Corg, δ13Corg, and lignin phenols to assess the relative contributions of petrogenic, soil-derived, plant-derived, and marine OC to the sedimentary OC pool. The analyzed fjord sediments consisted, on average, of 59.0 ± 28.1% petrogenic OC, 16.8 ± 12.1% soil-derived OC, 2.5 ± 2.2% plant-derived OC, and 21.8 ± 18.5% marine OC. This approach highlights the substantial contributions of petrogenic and aged soil-derived OC to present-day sedimentary OC in Svalbard fjords. Considering predicted global warming, accelerated inputs of petrogenic and soil-derived OC into fjords due to rapid glacier retreat may significantly impact the active carbon cycle and potentially contribute to CO2 emissions to the atmosphere, depending on burial efficiency.

Environmental toxicants--induced epigenetic alterations and their reversers.

Epigenetics has been emphasized in the postgenome era to clarify obscure health risks of environmental toxicants including endocrine disrupting chemicals (EDCs). In addition, mixed exposure in real life can modify health consequences of the toxicants. Particularly, some nutritional and dietary materials modify individual susceptibility through changes in the epigenome. Therefore, we focused on some environmental toxicants that induce epigenetic alterations, and introduced chemopreventive materials to reverse the toxicants-induced epigenetic alterations. Methodologically, we used global and specific DNA methylation as epigenetic end points and searched epigenetic modulators in food. We reviewed various epigenetic end points induced by environmental toxicants including alcohol, asbestos, nanomaterials, benzene, EDCs, metals, and ionizing radiation. The epigenetic end points can be summarized into global hypomethylation and specific hypermethylation at diverse tumor suppress genes. Exposure timing, dose, sex, or organ specificity should be considered to use the epigenetic end points as biomarkers for exposure to the epimutagenic toxicants. Particularly, neonatal exposure to the epimutagens can influence their future adult health because of characteristics of the epimutagens, which disrupt epigenetic regulation in imprinting, organogenesis, development, etc. Considering interaction between epimutagenic toxicants and their reversers in food, we suggest that multiple exposures to them can alleviate or mask epigenetic toxicity in real life. Our present review provides useful information to find new end points of environmental toxicants and to prevention from environment-related diseases.

Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes.

Metal ions existing in the environment could influence the estrogen pathway in aquatic animal, but the detailed mechanism is still delusive. We here showed that in male Carassius auratus hepatocytes, copper (Cu) or cadmium (Cd), did not directly induce vitellogenin (VTG) expression. Interestingly, co-exposure with Cd²âº (or Cu²âº) and 17-ß-estradiol (E2) greatly increased the VTG level, comparing with single treatment of E2. Meanwhile, Cd²âº or Cu²âº (but not E2) triggers HSP70 expression. But, mixture of Cd²âº or Cu²âº with E2 did not obviously raise HSP70 level. E2 also had no obvious effect on reactive oxygen species. Co-treatment of Cd²âº and E2 showed no obvious increase compared to single treatment with Cd²âº. We further assume that Cd²âº-involved oxidative stress generates misfolded proteins, resulting in the competition of HSP70 proteins from a heterocomplex (with estrogen receptor). Thus, dissociation of the heterocomplex actives the receptor-ligand binding activity and promotes the E2-induced VTG expression.

High-Dose Radiation-Induced Changes in Murine Small Intestinal Motility: Are the Changes in the Interstitial Cells of Cajal or in the Enteric Nervous System?

Murine small intestinal motility consists of phasic contraction from interstitial cells of Cajal (ICC) and migrating motor complexes (MMCs) from the enteric nervous system. The number of ICC is reduced in various gastrointestinal disorders, and this effect can be reversed once the disorder is resolved through cellular and tissue remodelling. Exposure to high-dose radiation can induce inflammation and alter intestinal motility. In this study, we investigated the changes in the small intestinal motility of 8- to 10-week-old male C3H/HeN mice after high-dose (13 Gy) irradiation. The aim of this study was to determine whether those changes are caused by changes in the ICC or enteric nervous system. After irradiation, the small intestine was dissected and stored in oxygenated Krebs-Ringer bicarbonate solution. The tension of contractions and intracellular membrane potentials were recorded at day 0, 1, 3 and 5 after irradiation and compared with those of sham-irradiated mice. Histological evaluation was performed by immunohistochemistry and apoptosis was evaluated. Quantitative real-time polymerase chain reaction (qPCR) for c-kit mRNA was also performed. Phasic contractions were not changed at day 0, 1, 3 and 5 after irradiation and did not significantly differ from those in the control mice. Slow waves were also sustained after irradiation. However, the frequency of migrating motor complexes (MMCs) was significantly higher at day 0 and 1 after exposure and the amplitude and area under the curve were significantly lower at day 3 after exposure compared with control mice. MMCs were recovered at day 5 with no difference from those of the control mice. ICC were detected after irradiation by immunohistochemistry for c-kit, and c-kit mRNA levels did not differ between sham-irradiated and irradiated mice. Histological evaluation showed that the most severe inflammation was detected at day 3 after irradiation, and apoptosis was detected only in the mucosa. Acetylcholine increased the contractility after irradiation, and tetrodotoxin decreased the number of MMCs in sham-irradiated and irradiated mice. N(w)-oxide-l-arginine (L-NA) increased the number of MMCs. MMCs were recovered after L-NA treatment at day 3 after irradiation. Sodium nitroprusside decreased the MMCs in sham-irradiated and irradiated mice. Exposure to high-dose radiation did not alter phasic contractions and slow waves in the small intestine of mice, which suggests that ICC and their functions may be sustained after high-dose irradiation. Mucosal inflammation was severe after irradiation and there were some changes in MMCs related to the enteric nervous system.

Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia).

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes, with clinical signs of severe ulcers and hemorrhages in the skeletal muscle. The internal transcribed spacer (ITS) region, which is widely used in taxonomy and molecular phylogeny because of a high degree of variation, was compared for 21 cloned strains of M. avidus (Ciliophora, Scuticociliatia). These strains were isolated from olive flounder Paralichthys olivaceus, ridged-eye flounder Pleuronichthys cornutus and spotted knifejaw Oplegnathus fasciatus in Korea and Japan. The ITS1 (140 bp), ITS2 (236 bp) and 5.8S (119 bp) regions from 21 strains were identical, indicating that these regions are highly conserved in M. avidus. Phylogenic analysis of ITS2 shows that the ciliate should be included in the Philasterida with a close relationship to Pseudocohnilembus hargisi. This study exhibits the first detailed analysis of the ITS1, 5.8 S and ITS2 rRNA regions of M. avidus.