Developmental toxicity of short-chain chlorinated paraffins on early-stage chicken embryos in a shell-less (ex-ovo) incubation system.

Short-chain chlorinated paraffins (SCCPs) are listed as a category of globally controlled persistent organic pollutants (POPs) by the Stockholm Convention in 2017. However, SCCP toxicity, particularly their developmental toxicity in avian embryos, has not been well studied. In this study, we observed the early development of chicken embryos (Gallus gallus domesticus) by applying a shell-less (ex-ovo) incubation system developed in our previous studies. After exposing embryos at Hamburger Hamilton stage (HHS) 1 to SCCPs (control, 0.1% DMSO; SCCPs-L, 200 ng/g; SCCPs-M, 2000 ng/g; SCCPs-H, 20,000 ng/g), we observed the development of embryos from the 3rd to 9th incubation day. Exposure to SCCPs-M and -H induced a significant reduction in survival, with an LD50 of 3100 ng/g on the 9th incubation day. Significant dose-dependent decreases in body length were observed from days 4-9. We also found that SCCPs-H decreased the blood vessel length and branch number on the 4th incubation day. Additionally, SCCPs-H significantly reduced the heart rate on the 4th and 5th incubation days. These findings suggest that SCCPs may have potential of developmental and cardiovascular toxicity during the early stages of chicken embryos. Quantitative PCR of the mRNA of genes related to embryonic development showed that SLC16A10 (a triiodothyronine transporter) level decreased in the SCCPs-H group, showing a significant positive correlation with the body length of embryos. THRA level, a thyroid hormone receptor, was significantly decreased in the SCCPs-H group, whereas that of DIO3 level, a deiodinase was significantly increased. These results suggest that SCCPs exposure induces developmental delays via the thyroxine signaling pathway. Analysis of thyroid hormones (THs) in blood plasma also indicated a significant reduction in thyroxine (T4) levels in the SCCPs-H group on the 9th incubation day of embryos. In conclusion, SCCPs induce developmental toxicity by disrupting thyroid functions at the early-life stage of chicken embryos.

Plastiome: Plastisphere-enriched mobile resistome in aquatic environments.

Aquatic microplastics (MPs) act as reservoirs for microbial communities, fostering the formation of a mobile resistome encompassing diverse antibiotic (ARGs) and biocide/metal resistance genes (BMRGs), and mobile genetic elements (MGEs). This collective genetic repertoire, referred to as the "plastiome," can potentially perpetuate environmental antimicrobial resistance (AMR). Our study examining two Japanese rivers near Tokyo revealed that waterborne MPs are primarily composed of polyethylene and polypropylene fibers and sheets of diverse origin. Clinically important genera like Exiguobacterium and Eubacterium were notably enriched on MPs. Metagenomic analysis uncovered a 3.46-fold higher enrichment of ARGs on MPs than those in water, with multidrug resistance genes (MDRGs) and BMRGs prevailing, particularly within MPs. Specific ARG and BMRG subtypes linked to resistance to vancomycin, beta-lactams, biocides, arsenic, and mercury showed selective enrichment on MPs. Network analysis revealed intense associations between host genera with ARGs, BMRGs, and MGEs on MPs, emphasizing their role in coselection. In contrast, river water exhibited weaker associations. This study underscores the complex interactions shaping the mobile plastiome in aquatic environments and emphasizes the global imperative for research to comprehend and effectively control AMR within the One Health framework.

Tris(2-chloroethyl) phosphate (TCEP) exposure inhibits the epithelial-mesenchymal transition (EMT), mesoderm differentiation, and cardiovascular development in early chicken embryos.

Tris(2-chloroethyl) phosphate (TCEP) is an organophosphorus flame retardant used worldwide and has been detected in the tissues and eggs of wild birds. Our previous study reported that exposure to TCEP induced developmental delay and cardiovascular dysfunction with attenuated heart rate and vasculogenesis in early chicken embryos. This study aimed to investigate the molecular mechanisms underlying the cardiovascular effects of TCEP on chicken embryos using cardiac transcriptome analysis and to examine whether TCEP exposure affects epithelial-mesenchymal transition (EMT) and mesoderm differentiation during gastrulation. Transcriptome analysis revealed that TCEP exposure decreased the expression of cardiac conduction-related genes and transcription factors on day 5 of incubation. In extraembryonic blood vessels, the expression levels of genes related to fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) were significantly reduced by TCEP exposure and vasculogenesis was suppressed. TCEP exposure also attenuated Snail family transcriptional repressor 2 (SNAI2) and T-box transcription factor T (TBXT) signaling in the chicken primitive streak, indicating that TCEP inhibits EMT and mesoderm differentiation during gastrulation at the early developmental stage. These effects on EMT and mesoderm differentiation may be related to subsequent phenotypic defects, including suppression of heart development and blood vessel formation.

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate.

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3-9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4-7. The somitic angle increased significantly on Days 4-6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Effects of exposure to oxytetracycline on the liver proteome of red seabream (Pagrus major) in a real administration scenario.

Oxytetracycline (OTC) is a widely used antibiotic in aquaculture. In this study, red seabream (Pagrus major), the most popular aquaculture species in Japan, were treated with OTC mimicking a real administration scenario in aquaculture. The treatment groups were as follows: no OTC, 40 mg/kg body wt/day (equivalent to the dose used in actual aquaculture), or 178 mg/kg body wt/day. The first exposure was conducted for a week (1st OTC exposure period), followed by a 4-week interval, and the second exposure was for one week (2nd OTC exposure period). We investigated the effects of OTC on the liver proteome with the isobaric tags for relative and absolute quantitation (iTRAQ) technology accompanied by liquid chromatography and mass spectrometry. The pathway and disease enrichment analyses of differentially abundant proteins in OTC-exposed groups compared to their respective controls showed that the abundance of proteins related to the immune and nervous systems was altered after the 1st and 2nd OTC exposures, respectively. Quantitative real-time PCR of the transcripts of immune-related genes corroborated with the results of proteome analysis. OTC exposure also modulated the expression of metabolism-related proteins after the 1st and 2nd OTC exposures. Furthermore, after four weeks of the 2nd exposure, weight loss and changes in the expression of proteins related to metabolism were observed, suggesting that OTC exposure disrupts the metabolic system and causes growth inhibition. Based on these results, we suggest that the use of OTC in aquaculture poses a health risk in fish species. Thus, we need to pay more attention to the contamination with OTC in aquaculture.

Effects of tris(2-chloroethyl) phosphate exposure on chicken embryos in a shell-less incubation system.

Tris(2-chloroethyl) phosphate (TCEP) is an organophosphate flame retardant that used in textiles, industrial materials, and furniture to delay the spread of fire after ignition. TCEP has been detected in the tissues and eggs of fish and birds. However, there are no studies regarding the effects of TCEP on avian embryos. In the present study, we investigated the developmental toxicity of TCEP exposure on chicken embryos in a shell-less incubation system, which enables in situ observation. Chicken embryos were treated with graded doses of TCEP (50, 250, and 500 nmol/g egg) on incubation day 0. The survival rate, morphological biometrics, heart rate, and length and branch number of extraembryonic blood vessels were measured on incubation days 3-9. Survival rates were reduced from incubation day 3 and were significantly decreased until day 9. Body length, head + bill length and eye diameter were significantly reduced by TCEP exposure. Regarding skeletal effects, spine length was decreased in a dose-dependent manner on day 9. Body weight on day 9 significantly reduced in all TCEP treatment groups. These results suggest that TCEP exposure to >50 nmol/g egg retards development in chicken embryos. TCEP exposure to 500 nmol/g egg significantly increased heart weight to body weight ratio in the embryos. More than 250 nmol/g egg of TCEP significantly reduced the heart rate of embryos in the early developmental stage. The formation of extraembryonic blood vessels and the number of erythrocytes were significantly reduced even with 50 nmol/g egg of TCEP. These findings suggest that TCEP exposure specifically affects the cardiovascular system in chicken embryos, which leads to developmental delay. The results of this study also demonstrate that the shell-less incubation system can be used to continuously monitor the effects of chemicals on developing avian embryos.

Tetracycline Resistance Gene Profiles in Red Seabream (Pagrus major) Intestine and Rearing Water After Oxytetracycline Administration.

Marine aquaculture fish and the environment are possible hot spots for the maintenance and spread of antibiotic resistance genes (ARGs). We here show the time courses of changes of six tetracycline resistance genes (tet) in fish rearing seawater and fish intestine in tank experiments. Experimental tanks were prepared as oxytetracycline (OTC) administration tanks and those without OTC. It was found that tet(B), tet(M), and tet(W) were dominant in seawater among the six tet genes. tet(B) and tet(M) abundances increased immediately after OTC administration, indicating that OTC served as a selective pressure to increase the proportion of tet-possessing bacteria. In contrast, the abundance of tet genes in the fish intestine did not differ between the with- and without-OTC administration groups, and clearly was not altered by OTC administration. Profile changing of tet in seawater and fish intestine did not synchronize. These observations suggested that the dynamics of intestinal tet-possessing bacteria do not directly reflect the environment, but reflect selection within the intestine.