Insight into the mechanisms of neuroendocrine toxicity induced by 6:2FTCA via thyroid hormone disruption.

6:2 fluorotonic carboxylic acid (6:2 FTCA), a novel substitute for perfluorooctanoic acid (PFOA), is being used gradually in industrial production such as coatings or processing aids, and its detection rate in the aqueous environment is increasing year by year, posing a potential safety risk to aquatic systems and public health. However, limited information is available on the effects and mechanism of 6:2 FTCA. Therefore, this study was conducted to understand better the neuroendocrine effects of early exposure to 6:2 FTCA and the underlying mechanisms on zebrafish. In this study, zebrafish embryos were treated to varied doses of 6:2 FTCA (0, 0.08 µg/mL, 0.8 µg/mL and 8 µg/mL) at 4 h post-fertilization (hpf) for a duration of six days, which exhibited a pronounced inhibition of early growth and induced a disorganized swim pattern characterized by reduced total swim distance and average swim speed. Simultaneously, the thyroid development of zebrafish larvae was partially hindered, accompanied by decreased T3 levels, altered genes associated with the expression of thyroid hormone synthesis, transformation and transportation and neurotransmitters associated with tryptophan and tyrosine metabolic pathways. Molecular docking results showed that 6:2 FTCA has a robust binding energy with the thyroid hormone receptor (TRß). Moreover, exogenous T3 supplementation can partially restore the adverse outcomes. Our findings indicated that 6:2 FTCA acts as a thyroid endocrine disruptor and can induce neuroendocrine toxic effects. Furthermore, our results show that targeting TRß may be a potentially therapeutic strategy for 6:2 FTCA-induced neuroendocrine disrupting effects.

A three-plasma miRNA panel predicts the risk of colorectal cancer: a community-based nested case‒control study.

Circulating microRNAs (miRNAs) have been considered potential markers for the early detection of malignant colorectal cancer (CRC). We aimed to identify a group of miRNAs for the early detection of CRC and assess their predictive ability in a community-based population in China. A nested case‒control study consisting of 97 incident colorectal cancer cases and 103 frequency-matched healthy controls was conducted. The data were randomly assigned into a training set (60%) and a test set (40%). We selected and detected 10 kinds of miRNAs in plasma samples. Multivariate logistic regression analysis was used to identify miRNAs associated with colorectal cancer risk in the training set and test set. Then, we evaluated the predictive ability of the identified miRNAs by the receiver operating characteristic curve (ROC). In this study, three miRNAs (miRNA-29a, miRNA-125b, miRNA-145) were significantly associated with colorectal cancer risk in both the training set and test set. The sensitivity of the identified miRNAs ranged from 0.854 to 0.961. After adding the identified miRNAs, the AUC (area under the curve) value significantly increased from 0.61 to 0.71 compared with the basic model consisting of only basic demographic information. We identified a three-plasma miRNA signature that may serve as a novel non-invasive biomarker in early CRC detection and in predicting individual CRC risk in the generation population.

Developmental toxicity of disinfection by-product monohaloacetamides in embryo-larval stage of zebrafish.

As an emerging class of nitrogenous disinfection by-products (N-DBPs), haloacetamides (HAcAms) have been widely detected in drinking water. Limited toxicity studies have shown an inconsistent toxicity of monoHAcAms, including CAcAm, BAcAm and IAcAm. In this study, the developmental toxicity of monoHAcAms was evaluated in embryo-larval stage of zebrafish. Embryos were exposed to one concentration of 2.50, 5.00, 10.0, 20.0, 40.0 and 80.0 mg/L monoHAcAms from 4 h post-fertilization (hpf) to 120 hpf. Multiple endpoints, including hatching rate, morphological abnormalities, mortality as well as locomotor behavior were assessed at specified stages (24, 48, 72, 96 and 120 hpf). Results showed that 80 mg/L CAcAm and 40 mg/L BAcAm significantly decreased the hatching rate, IAcAm decreased the hatching rate and delayed the hatching process in a concentration-dependent manner with an EC50 of 16.37 mg/L at 72 hpf. The frequency and severity order of morphological abnormalities increased with the raised exposure concentrations and prolonged exposure time, and the corresponding EC50 at 96 hpf were 21.10, 9.77 and 16.60 mg/L for CAcAm, BAcAm and IAcAm, respectively. MonoHAcAms exposure resulted in a time- and dose-dependent response in mortality and the calculated LC50 at 72 hpf were 38.44, 17.74 and 28.82 mg/L for CAcAm, BAcAm and IAcAm, respectively. Based on EC50 for morphological abnormalities and LC50, a toxicity rank order of BAcAm > IAcAm > CAcAm was observed. Different degrees of hyperactivity and hypoactivity were observed from locomotor behavior analysis in larvae from ≤10.0 mg/L monoHAcAms exposure groups. The light-dark periodic change was disappeared in larvae of 10.0 mg/L BAcAm exposure group. In summary, our study showed that monoHAcAms were developmentally toxic to zebrafish even at very low concentrations and BAcAm exerted higher toxicity than IAcAm and CAcAm. These results will further our understanding of the toxicity of HAcAms and its potential toxicological impact on human and ecological environment.

Different cytotoxicity of disinfection by-product haloacetamides on two exposure pathway-related cell lines: Human gastric epithelial cell line GES-1 and immortalized human keratinocyte cell line HaCaT.

Humans are exposed to disinfection by-products (DBPs) mainly through drinking water ingestion and dermal contact. As an emerging class of nitrogenous DBPs (N-DBPs), haloacetamides (HAcAms) have been found to have significantly higher cytotoxicity than regulated DBPs. In this study, we investigated the cytotoxicity of HAcAms on two exposure pathway-related cell lines: human gastric epithelial GES-1 cells and immortalized keratinocytes HaCaT. Our results showed that the ranking order of cytotoxicity of 13 HAcAms was different between HaCaT and GES-1 cells. In addition, the 50% inhibitive concentration in HaCaT was 1.01-3.29 times that in GES-1. Further comparison among GES-1, HaCaT and CHO cell lines confirmed that different cell lines exhibited different sensitivity to the same compound. Importantly, HAcAms showed 5.83-7.13 × 104 times higher toxicity than the well-clarified DBP chloroform, clearly demonstrating the increased toxicity of HAcAms. Finally, using a novel high-content screening (HCS) analysis, we found that 39.29% of chlorinated HAcAms, 42.86% of brominated HAcAms and 16.07% of iodinated HAcAms significantly affected at least one of the cell-health parameters, such as nuclear size, membrane permeability, mitochondrial membrane potential, or cytochrome c release, in GES-1 or HaCaT cells. Thus, brominated HAcAms appear to have stronger effects under the sublethal exposure dose, possibly causing cytotoxicity via apoptosis. Together, our study provides new insights to the toxicity of HAcAms and a comprehensive toxicology dataset for health risk assessment.

Simultaneous determination of three azo dyes in food product by ion mobility spectrometry.

Color is an important property for food evaluation. Synthetic azo dyes are usually used in food product to obtain better appearance because of their stability and low cost. However, such dyes should be strictly controlled because of their potential threat to human health. A simple, rapid and sensitive method has been developed to determine orange II, allura red, and para red simultaneously by ion mobility spectrometry. The three dyes could be separated at the same time and the migration time of orange II, allura red, and para red are 12.070±0.010, 8.180±0.015, and 11.037±0.016ms, respectively. The effects of different parameters, such as pH, solvent, percentage of water, were investigated to establish the optimal condition. The detection limits were 0.1, 0.05, and 0.2µg/mL for orange II, allura red, and para red, respectively. The recoveries of the three azo dyes from jellies were all higher than 81%. The developed method is fast and accurate for the detection of the three synthetic dyes.

Lead exposure increases blood pressure by increasing angiotensinogen expression.

Lead exposure can induce increased blood pressure. Several mechanisms have been proposed to explain lead-induced hypertension. Changes in angiotensinogen (AGT) expression levels or gene variants may also influence blood pressure. In this study, we hypothesized that AGT expression levels or gene variants contribute to lead-induced hypertension. A preliminary HEK293 cell model experiment was performed to analyze the association between AGT expression and lead exposure. In a population-based study, serum AGT level was measured in both lead-exposed and control populations. To further detect the influence of AGT gene single nucleotide polymorphisms (SNPs) in lead-induced hypertension, two SNPs (rs699 and rs4762) were genotyped in a case-control study including 219 lead-exposed subjects and 393 controls. Lead exposure caused an increase in AGT expression level in HEK 293 cell models (P < 0.001) compared to lead-free cells, and individuals exposed to lead had higher systolic and diastolic blood pressure (P < 0.001). Lead-exposed individuals had higher serum AGT levels compared to controls (P < 0.001). However, no association was found between AGT gene SNPs (rs699 and rs4762) and lead exposure. Nevertheless, the change in AGT expression level may play an important role in the development of lead-induced hypertension.

Extracellular dGMP enhances Deinococcus radiodurans tolerance to oxidative stress.

Free extracellular DNA provides nutrition to bacteria and promotes bacterial evolution by inducing excessive mutagenesis of the genome. To understand the influence of extracellular DNA fragments on D. radiodurans, we investigated cell growth and survival after extracellular DNA or dNMPs treatment. The results showed that the extracellular DNA fragments inhibited the growth of D. radiodurans. Interestingly, dGMP, a DNA component, enhanced D. radiodurans tolerance to H(2)O(2) and gamma-radiation significantly. Further experiments indicated that extracellular dGMP stimulated the activity of one catalase (KatA, DR1998), and induced gene transcription including the extracellular nuclease (drb0067). When this only extracellular nuclease gene (drb0067) in D. radiodurans was deleted, the mutant strain showed more sensitive to H(2)O(2) and gamma-radiation than the wild type strain. These results suggest that DRB0067 plays an important role in oxidative stress resistance. Taken together, we proposed a new anti-oxidation mechanism in D. radiodurans. This mechanism acts to increase expression levels of DRB0067 which then secretes active nuclease to degrade extracellular DNA fragments. The extracellular nuclease has a two-fold benefit, creating more free dNTPs for further cell protection and the removal of extracellular DNA fragments.

Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans.

The high intracellular Mn/Fe ratio observed within the bacteria Deinococcus radiodurans may contribute to its remarkable resistance to environmental stresses. We isolated DR2539, a novel regulator of intracellular Mn/Fe homeostasis in D. radiodurans. Electrophoretic gel mobility shift assays (EMSAs) revealed that DR2539 binds specifically to the promoter of the manganese acquisition transporter (MntH) gene, and that DR0865, the only Fur homologue in D. radiodurans, cannot bind to the promoter of mntH, but it can bind to the promoter of another manganese acquisition transporter, MntABC. ß-galactosidase expression analysis indicated that DR2539 acts as a manganese- and iron-dependent transcriptional repressor. Further sequence alignment analysis revealed that DR2539 has evolved some special characteristics. Site-directed mutagenesis suggested that His98 plays an important role in the activities of DR2539, and further protein-DNA binding activity assays showed that the activity of H98Y mutants decreased dramatically relative to wild type DR2539. Our study suggests that D. radiodurans has evolved a very efficient manganese regulation mechanism that involves its high intracellular Mn/Fe ratio and permits resistance to extreme conditions.

Function and biochemical characterization of RecJ in Deinococcus radiodurans.

The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.

Repression of recA induction by RecX is independent of the RecA protein in Deinococcus radiodurans.

Besides inhibiting RecA activity at the protein level, Deinococcus radiodurans RecX can suppress RecA induction at the transcriptional level. The regulation of RecX on recA induction is independent of RecA activity, and its N terminus is involved in this process.

A novel carotenoid 1,2-hydratase (CruF) from two species of the non-photosynthetic bacterium Deinococcus.

A novel carotenoid 1,2-hydratase (CruF) responsible for the C-1',2' hydration of gamma-carotene was identified in the non-photosynthetic bacteria Deinococcus radiodurans R1 and Deinococcus geothermalis DSM 11300. Gene expression and disruption experiments demonstrated that dr0091 and dgeo2309 encode CruF in D. radiodurans and D. geothermalis, respectively. Their homologues were also found in the genomes of cyanobacteria, and exhibited little homology to the hydroxyneurosporene synthase (CrtC) proteins found mainly in photosynthetic bacteria. Phylogenetic analysis showed that CruF homologues form a separate family, which is evolutionarily distant from the known CrtC family.

[Cloning, expression and radiation inducibility of RadA from the hyperthermophilic archaeon Sulfolobus tokodaii].

RecA/Rad51/RadA recombinases are important recombination proteins with conserved functions. Studies on the enzymes have shown that members of RecA/Rad51/RadA family from bacteria, eukaryota, methanogens and halophilic archaea have UV inducibility. However, the UV inducibility of RadA homologues from hyperthermophilic archaea is controversial. We analyzed the UV inducibility of Sulfolobus tokodaii RadA by RT-PCR and immune assays. Comparing with the mock, the transcription and expression of the radA increased 2 and 1.5 folds respectively after UV irradiation at 100 J/m2, or 3 and 1 fold at 200 J/m2. These results demonstrated that S. tokodaii RadA could be induced after UV treatment. In addition, proteome induction analysis proved that there existed a DNA damage induction response in S. tokodaii, which further supported RadA inductility in this hyperthermophilic archaeon.

Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii.

ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.