Quantitative determination of bromochloroacetamide in mice urine by gas chromatography combined with salting-out assisted dispersive liquid-liquid microextraction.

Bromochloroacetamide (BCAcAm) is the main haloacetamide (HAcAm) detected in drinking water in different regions and exhibits strong cytotoxicity and genotoxicity. However, there is no appropriate method for detecting BCAcAm in urine or other biological samples, and thus, the internal exposure level in the population cannot be accurately assessed. In this study, a gas chromatography-electron capture detector (GC-ECD) was combined with salting-out assisted dispersive liquid-liquid microextraction (SA-DLLME) to develop a rapid and robust method for BCAcAm detection in urine of mice continuously exposed to BCAcAm. The factors influencing the pre-treatment procedure, including the type and volume of extraction and disperser solvents, extraction and standing time, and the amount of salt, were evaluated systematically. Under the optimised conditions, the analyte achieved good linearity in the spiked concentration range of 1.00-400.00 µg L-1, and the correlation coefficient was higher than 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.17 µg L-1 and 0.50 µg L-1, respectively. The recoveries ranged from 84.20% to 92.17%. The detection of BCAcAm at three different calibration levels using this method afforded an intra-day precision of 1.95-4.29%, while the inter-day precision range was 5.54-9.82% (n = 6). This method has been successfully applied to monitor the concentration of BCAcAm in mouse urine in toxicity experiments and can provide technical support for assessing human internal exposure levels and health risks in later studies.

Rumen microbiota-host transcriptome interaction mediates the protective effects of trans-10, cis-12 CLA on facilitating weaning transition of lambs.

Developing alternatives to antibiotics for prevention of gastrointestinal dysbiosis in early-weaning farmed animals is urgently needed. This study was to explore the potential effects of trans-10, cis-12 conjugated linoleic acid (CLA) on maintaining ruminal homeostasis of young ruminants during the weaning transition period. Thirty neonatal lambs were selected (6 lambs per group) and euthanized for rumen microbial and epithelial analysis. The lambs were weaned at 28 d and experienced the following 5 treatments: euthanized on d 28 as the pre-weaning control (CON0), fed starter feed for 5 (CON5) or 21 (CON21) d, fed starter feed with 1% of CLA supplemented for 5 (CLA5) or 21 (CLA21) d. Results showed that the average daily weight gain and dry matter intake were significantly higher in CLA5 than CON5 group. As compared with the CON5 and CON21 group, the relative abundances of volatile fatty acid (VFA) producing bacteria including Bacteroides, Treponema, Parabacteroides and Anaerovibrio, as well as the concentrations of acetate, butyrate and total VFA were significantly increased in CLA5 and CLA21 group, respectively. Integrating microbial profiling and epithelial transcriptome results showed that 7 downregulated inflammatory signaling-related host genes IL2RA, CXCL9, CD4, CCR4, LTB, SPP1, and BCL2A1 with CLA supplementation were significantly negatively correlated with both VFA concentration and VFA producing bacteria, while 3 (GPX2, SLC27A2 and ALDH3A1) and 2 (GSTM3 and GSTA1) upregulated metabolism-related genes, significantly positively correlated with either VFA concentration or VFA producing bacteria, respectively. To confirm the effects of CLA on epithelial signal transduction, in vitro experiment was further conducted by treating rumen epithelial cells without or with IL-17A + TNF-α for 12 h after pretreatment of 100 µM CLA or not (6 replicates per treatment). The results demonstrated the anti-inflammatory effect of CLA via suppressing the protein expression of NF-кB p-p65/p65 with the activation of peroxisome proliferator-activated receptor gamma (PPARγ). In conclusion, CLA supplementation enhanced the ruminal microbiota-driven transcriptional regulation in healthy rumen epithelial development via rumen VFA production, and CLA may therefore serve as an alternative way to alleviate early-weaning stress and improve physiological and metabolic conditions of young ruminants.

Pyridoxal 5'-phosphate alleviates prenatal pyridaben exposure-induced anxiety-like behaviors in offspring.

Pyridaben (PY) is a widely used organochlorine acaricide, which can be detected in the peripheral blood of pregnant women. Available evidence suggests that PY has reproductive toxicity. However, it remains uncertain whether prenatal PY exposure impacts neurobehavioral development in offspring. Here, we administered PY to pregnant mice at a dose of 0.5 and 5 mg kg-1 day-1 via gavage and observed anxiety-like behaviors in PY offspring aged five weeks. We then integrated the metabolome and transcriptome of the offspring's brain to explore the underlying mechanism. Metabolome data indicated that the vitamin B6 metabolism pathway was significantly affected, and the pyridoxal 5'-phosphate (PLP) concentration and the active form of vitamin B6 was significantly reduced. Moreover, the transcriptome data showed that both PLP generation-related Pdxk and anxiety-related Gad1 were significantly down-regulated. Meanwhile, there was a decreasing trend in the concentration of GABA in the hippocampal DG region. Next, we supplemented PLP at a dose of 20 mg kg-1 day-1 to the PY offspring via intraperitoneal injection at three weeks. We found up-regulated expression of Pdxk and Gad1 and restored anxiety-like behaviors. This study suggests that prenatal exposure to PY can disrupt vitamin B6 metabolism, reduce the concentration of PLP, down-regulate the expression levels of Pdxk and Gad1, inhibit the production of GABA, and ultimately lead to anxiety-like behaviors in offspring.

Effects of glufosinate-ammonium on male reproductive health: Focus on epigenome and transcriptome in mouse sperm.

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its neural and reproductive toxicity. To uncover potential effects of GLA on male reproductive health in mammals, adult male C57BL/6J mice were administered 0.2 mg/kg·d GLA for 5 weeks. After examination on fertility, testis histology and semen quality in the GLA group, we performed deep sequencing to identify repressive epigenetic marks including DNA methylation and histone modifications (H3K27me3 and H3K9me3), together with mRNA transcript levels in sperm. Then, we integrated multi-omics sequencing data to comprehensively explore GLA-induced epigenetic and transcriptomic alterations. We found no significant difference either on fertility, testis histology or semen quality-related indicators. As for epigenome, the protein level of H3K27me3 was significantly increased in GLA sperm. Next generation sequencing showed alterations of these epigenetic marks and extensive transcription inhibition in sperm. These differential repressive marks were mainly distributed at intergenic regions and introns. According to results by Gene Ontology enrichment analysis, both differentially methylated and expressed genes were mainly enriched in pathways related to synapse organization. Subtle differences in genomic imprinting were also observed between the two groups. These results suggested that GLA predominantly impaired sperm epigenome and transcriptome in mice, with little effect on fertility, testis histology or semen quality. Further studies on human sperm using similar strategies need to be conducted for a better understanding of the male reproductive toxicity of GLA.

Evaluation of atrazine neurodevelopment toxicity in vitro-application of hESC-based neural differentiation model.

Atrazine is one of the widely used herbicides in the world and most of the current researches on atrazine neurodevelopment toxicity have focused on rodents or zebrafish models in vivo, resulting in relatively high cost, time consumption, and lower translational value to identify its hazard for the developing brain. Major international initiatives have pushed forward to convert the traditional animal-based developmental toxicity tests to in vitro assays using human cells to detect and predict chemical health hazards. In this study, we presented a human neural differentiation model based on human embryonic stem cells (hESC) that can be used to test toxicity at different stages of neural differentiation in vitro. hESC were differentiated into neural stem cells (NSC) and then terminally differentiated towards mixed neurons and glial cells for 21 days. Cell survival, proliferation, cell cycle, apoptosis, and gene expression levels were examined. Our results demonstrated that atrazine inhibited the proliferation of hESC and NSC, and showed different toxic sensitivity on these two kinds of cells. Also, atrazine blocked the NSC cell cycle G1 phase via down-regulating CCND1, CDK2, and CDK4, with no obvious effect on apoptosis. In addition, atrazine curbed EB spontaneous differentiation and NSC-induced neurons and glia cells differentiation. Atrazine altered genes expression levels of PAX6, TUBB3, NCAM1, GFAP, TH, NR4A1, and GRIA1. From the data we obtained, we recognized that the dopaminergic system was not the only target of atrazine neurotoxicity, glutamatergic neurons and astrocytes were also adversely affected.

Effects of exposure to urban particulate matter SRM 1648a during pregnancy on the neurobehavioral development of offspring mice.

The development of the nervous system is crucial to a child's health. However, the nervous system is also susceptible to a variety of factors during development. To date, epidemiological studies have reported controversial results on the relationship between prenatal exposure to particulate matter (PM) and neurobehavioral development. Thus, we investigated the effect of PM exposure during pregnancy on the neurobehavioral development of offsprings. Adult C57BL/6 mice were exposed to PM from gestation day (GD) 0.5-21 by the intratracheal instillation. The daily exposure doses were 250 µg/kg.b.w and 2500 µg/kg.b.w respectively. The offspring mice began behavioral tests at the 5th week. We assessed neurobehavioral development, and the gene expression level changes in the mouse hippocampus using RNA-seq. In the open field test, the movement distance in the central area was significantly decreased in the high-dose group. Serum free triiodothyronine (FT3) levels were significantly increased in male offspring mice with prenatal high-dose PM exposure. The RNA-seq results suggested that the Prkca, Med12l, Ep300, and Slc16a10 in the thyroid hormone signaling pathway were significantly decreased in offspring mice in the high-dose group. Our data showed that prenatal PM exposure caused the offspring mice's anxiety-like behaviors and increased serum FT3 levels. The changes in thyroid hormone pathway-related genes might be the causes of the above series of changes.

Glufosinate-Ammonium Induced Aberrant Histone Modifications in Mouse Sperm Are Concordant With Transcriptome in Preimplantation Embryos.

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its male reproductive toxicity. Abnormalities in sperm histone modification induced by GLA exposure observed in our previous study aroused our interest in whether such alterations could further affect embryonic gene expression. Here we administered adult male mice with 0.2 mg/kg⋅day of GLA for 5 weeks to collect their sperm or 4-cell embryos after copulation. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing showed alterations of sperm H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are active histone modification marks involved in embryo development, while RNA sequencing identified differentially expressed genes in 4-cell embryos. Differentially H3K4me3 and H3K27ac occupied regions were mainly distributed at the gene promoters and putative enhancers, and were enriched in pathways related to the immune system and nervous system. Integrative analysis of these sequencing data showed that genes such as Mgl2 with increased H3K4me3 and H3K27ac in sperm were up-regulated in embryos, and vice versa for genes such as Dcn. Additionally, differentially occupied H3K4me3 and H3K27ac in sperm were linked to gene expression changes in both paternal and maternal alleles of 4-cell embryos. In conclusion, GLA-induced changes in sperm H3K4me3 and H3K27ac are concordant with gene expression in preimplantation embryos, which might further affect embryo development and offspring health.

Metabolomics Reveals Metabolic Changes Caused by Low-Dose 4-Tert-Octylphenol in Mice Liver.

Background: Humans are constantly exposed to low concentrations of 4-tert-octylphenol (OP). However, studies investigating the effects of low-dose OP on the liver are scarce, and the mechanism of these effects has not been thoroughly elucidated to date. Methods: Adult male institute of cancer research (ICR) mice were exposed to low-dose OP (0, 0.01 and 1 µg/kg/day) for 7 consecutive days. Weights of mice were recorded daily during the experiment. Blood serum levels of OP, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined, and haematoxylin-eosin (HE) staining of the liver was performed. We applied an integrated metabolomic and enzyme gene expression analysis to investigate liver metabolic changes, and the gene expression of related metabolic enzymes was determined by real-time PCR and ELISA. Results: OP in blood serum was increased after OP exposure, while body weights of mice were unchanged. Liver weight and its organ coefficient were decreased significantly in the OP (1 µg/kg/day) group, but ALT and AST, as well as the HE staining results, were unchanged after OP treatment. The levels of cytidine, uridine, purine and N-acetylglutamine were increased significantly, and the level of vitamin B6 was decreased significantly in mice treated with OP (1 µg/kg/day). The mRNA and protein levels of Cda and Shmt1 were both increased significantly in OP (1 µg/kg/day)-treated mice. Conclusions: Through metabolomic analysis, our study firstly found that pyrimidine and purine synthesis were promoted and that N-acetylglutamine was upregulated after low-dose OP treatment, indicating that the treatment disturbed nucleic acid and amino acid metabolism in mice liver.