Junlin Yuan

Effects of silica nanoparticles on growth and photosynthetic pigment contents of Scenedesmus obliquus

To assess the aquatic ecosystem safety for silica (SiO2) nanoparticles (NPs), the growth inhibition and photosynthetic pigment contents of Scenedesmus obliquus in logarithm growth phase exposed to SiO2 NPs and SiO2 bulk particles (BPs) suspensions were measured. SiO2 NPs with 10-20 nm diameters were found to be toxic. The 20% effective concentration (EC20) values for 72 and 96 hr were 388.1 and 216.5 mg/L, respectively. The contents of chlorophyll decreased significantly under moderate and high concentration (50, 100, and 200 mg/L) of SiO2 NPs after 96-hr exposure, but the carotenoids did not. SiO2 BPs were found to be nontoxic up to 200 mg/L. The toxicity of SiO2 NPs probablely due to their sorption to algal cells surface. The results imply that there is potential harm to aquatic environment by using SiO2 NPs, and it should deserve special concern.

Approach to distribution and accumulation of dibutyl phthalate in rats by immunoassay

Dibutyl phthalate (DBP) is mainly taken up by the general population from food intake. To estimate intake of phthalates, determining distribution and accumulation of DBP in biological materials was a critical need. In this work, we set up two novel approaches with a monoclonal antibody specific to DBP to determine the distribution and accumulation of DBP in vivo. The contents of DBP in liver, kidney, stomach and testes were detected by immunofluorescence assays and indirect competitive ELISA. This data give directly evidence that indicates the distribution and accumulation of DBP in vivo. Double-label immunofluorescence assay provides with a visual approach to determination of the distribution and accumulation of DBP. It indicated that DBP accumulated in subcutaneous tissue such as sweat gland, hair follicle. Both of immunofluorescence assay and ELISA can be used to detect the content of DBP in biological materials. Our assays showed that DBP accumulated in viscera being rich in fat, such as liver, kidney and could overcome physiological barriers to penetrate testes. The date suggested that the accumulations of DBP exposed through dermal route were less than that of oral route and most of DBP was metabolized in 2 or 3 days.

An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

Background Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. Methodology/Principal Findings A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. Conclusions/Significance The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.

Neurobehavioral changes induced by di(2-ethylhexyl) phthalate and the protective effects of vitamin E in Kunming mice

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in PVC that may leach into the environment, and has been shown to adversely affect the health of humans and animals. We undertook a study to ascertain the neurotoxicity of DEHP in Kunming mice. This study included three rounds of testing. In the first round, Kunming mice were exposed to different concentrations of DEHP (0, 5, 50, 500 mg kg−1 per day) after which their cognitive ability was assessed using the Morris water maze (MWM) test. The reactive oxygen species (ROS) content in tissue and the malondialdehyde (MDA) content of brains were also measured. In the second round, vitamin E (50 mg kg−1 per day) was given daily as an anti-oxidant via the intragastric route. Cognitive deficits and locomotor activity, as well as ROS and MDA contents were tested employing the same methods. In the third round, the depressive mood of mice after DEHP exposure (500 mg kg−1 per day) was measured using the open field test, the tail suspension test, and the forced swim test. The main findings of this study include: (1) a statistical association exists between DEHP oral exposure and spatial learning (DEHP 500 mg kg−1 per day) and memory (DEHP 50 mg kg−1 per day) dysfunction as ascertained by an MWM test of Kunming mice. (2) A statistical association was also found between DEHP oral exposure (50 and 500 mg kg−1 per day) and oxidative stress (ROS and MDA) of mouse brain tissue. (3) Co-administration of vitamin E (50 mg kg−1 per day) diminishes the elevation of ROS and MDA induced by DEHP (50 mg kg−1 per day) from significant levels to non-significant levels. (4) Co-administration of vitamin E (50 mg kg−1 per day) protects against mouse memory dysfunction induced by DEHP (50 mg kg−1 per day) from being significant to being not significant. (5) In the 5 mg kg−1 per day DEHP exposure groups, oxidative stress in brain tissue, and neurobehavioral changes were not found. (6) High dose DEHP exposure (500 mg kg−1 per day) may induce behavioral despair in mice. Conclusions: These data suggest that DEHP is neurotoxic with regard to cognitive ability and locomotor activity.

In vitro study on cytotoxicity and intracellular formaldehyde concentration changes after exposure to formaldehyde and its derivatives.

HeLa cells were exposed to formaldehyde and its metabolic derivatives, methanol, formic acid, and acetaldehyde, to investigate that the toxicity of formaldehyde is not caused by the chemical group. After 1 h of treatment with formaldehyde, mitochondrial assays showed that low concentrations (e.g. 10 μmol/L) of formaldehyde promoted growth of the HeLa cells, while higher concentrations (e.g. ≥62.5 μmol/L) inhibited cell growth; while all four chemicals at a concentration of 125 μmol/L affected cell growth, formaldehyde affected the largest. Reactive oxygen species concentration increased with the concentration of the exposure chemical. The endogenous formaldehyde content increased the most in the formaldehyde group, but in other three groups, it did not increase as the exposure concentration increased. Expression of dehydrogenase (formaldehyde dehydrogenase (FDH)) in the formaldehyde (10.40) and methanol (10.60) groups increased significantly compared with the control (1), while it was similar to the control in formic acid (0.90) and acetaldehyde (1.10) groups. Our results suggest that formaldehyde could affect cell activity and even enter cells. Exposure to formaldehyde changes the endogenous formaldehyde concentration in cells within 24 h, and this induces expression of FDH for formaldehyde degradation to maintain the formaldehyde balance. The toxicity of formaldehyde is not caused by the carbon atoms in the aldehyde, hydroxyl, or carboxyl groups. Formaldehyde is hypothesized to be an important signaling molecule in the regulation of cell growth and maintenance of the endogenous formaldehyde level.

Formaldehyde-induced paxillin-tyrosine phosphorylation and paxillin and P53 downexpression in Hela cells.

Abstract Formaldehyde (FA) is an environmental pollutant and an endogenous product believed to be involved in tumorigenesis. However, the underlying mechanism of observed FA effects has not been clearly defined. Paxillin is a focal adhesion protein that may play an important role in several signaling pathways. Many paxillin-interacting proteins are involved in the regulation of actin cytoskeleton organization, which is necessary for cell motility events associated with diverse biological responses, such as embryonic development, wound repair and tumor metastasis. P53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. In this study, we investigated the effects of FA on paxillin–tyrosine phosphorylation and P53 expression in Hela cells by Western blot and immunofluorescence. Western blot analysis revealed that nonlethal concentrations of FA (0.5, 1.0 and 2.0 mM, with the exposure time for 0.5, 1.0 and 2.0 h, respectively) had downregulated paxillin and wild-type p53 genes expression while upregulated paxillin–tyrosine phosphorylation significantly. At the same time, phosphotyrosine at the focal adhesion sites detected by immunofluorescence assay obviously increased in Hela cells incubated with 2.0 mM FA for 2 h. The results suggested that paxillin and p53 genes expression may be involved in FA-related adverse effects and the mechanism may be involved in paxillin–tyrosine phosphorylation.

Anti-bensulfuron methyl monoclonal antibody production and BSM-detecting indirect competitive enzyme-linked immunoassay establishment

Bensulfuron methyl (BSM) is widely used for agricultural purposes and has raised health concerns, as well as ecological problems. Immunoassay would be one of the most advantaged measurements compared with traditional methods for BSM detection and measurement. In order to develop indirect competitive enzyme-linked immunoassay (icELISA), the anti-BSM monoclonal antibody (anti-BSM MAb) was produced. For the MAb production, BSM was conjugated to OVA and injected to mice with Freuds adjuvant for immunisation. Antiserum screening has revealed successful immunising. One stable hybridoma cell line (2H1) was obtained through cell fusion between spleen cells of immunised mouse and SP 2/0 cells. The MAb secreted by 2H1 cells was of high affinity and sensitivity, as well as specificity to BSM. Then the protocol of the icELISA and standard curve for BSM measurement was made and examined by controlled application. Significantly, the application has exhibited 96.530%–107.2% recovery of BSM. The produced MAb and established immunoassay may facilitate the measurement of BSM and herein help to ensure food safety and regulate environmental protection.

Development of two enzyme-linked immunosorbent assay formats for thifluzamide residues’ analysis based on distinct polyclonal antibodies

ABSTRACT Haptens 2-Methyl-4-(trifluoromethyl)thiazole-5-carboxylic acid and 2,6-Dibromo-4-(trifluoromethoxy)aniline, the two moieties of thifluzamide, were conjugated with carrier proteins for the synthesis of artificial antigens. Two distinct anti-thifluzamide polyclonal antibodies (PAb-1 and PAb-2) were produced from the immunized female Balb/c mice. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) in two formats based on the PAbs was developed for thifluzamide analysis. The concentration of 50% inhibition (IC50) of ELISA-1 was 1.39 mg L−1 and its limit of detection (LOD) was 0.082 mg L−1. Meanwhile, ELISA-2 had a similar IC50 of 1.96 mg L−1 and a LOD of 0.074 mg L−1 as ELISA-1. Both the raised PAbs exhibited high specificity to thifluzamide. The recoveries for spiked samples including water and wheat ranged from 72.0% to 128.4%, and the accuracy of ELISA was confirmed by high-performance liquid chromatography. In summary, the ic-ELISA might be a promising tool for simple, sensitive and rapid detection of thifluzamide residues in real samples.

Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

ABSTRACT Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post‐exposure stage is crucial for a deep understanding of FA‐induced hematotoxicity. BALB/c mice were exposed to 3 mg/m3 FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post‐exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co‐exposure occurs frequently. After 7‐day recovery, colonies of progenitors for CFU‐GM and BFU‐E, and nucleated bone marrow cells in FA‐exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in CFU‐GM and BFU‐E from FA‐exposed mice were higher than controls, although the increase in 8‐OHdG was not significant. Granulocyte‐macrophage colony stimulating factor (GM‐CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA‐exposed mice respond to a small amount of GM‐CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co‐exposure with BZ was more potent for promoting CFU‐GM formation and inducing ROS in BFU‐E and 8‐OHdG in CFU‐GM during the post‐exposure period. The compensation of myeloid progenitors with elevated ROS and 8‐OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co‐exposure may pose a greater leukemia risk. HIGHLIGHTSNucleated bone marrow cell count recovered after 7 days post‐FA and/or BZ exposure.CFU‐GM showed an increase in colonies and 8‐OHdG after 7 days post‐FA + BZ exposure.Levels of ROS in CFU‐GM and BFU‐E were increased by FA or FA + BZ during recovery.Levels of GM‐CSF and EPOR were suppressed after 7 days post‐FA or FA + BZ exposure.Co‐exposure was more potent for some endpoints and may pose a greater leukemia risk.

Mono-butyl phthalate-induced mouse testis injury is associated with oxidative stress and down-regulated expression of Sox9 and Dazl

Mono-butyl phthalate (MBP) has reproductive toxicity but the related mechanisms have not been fully elucidated in vivo. We exposed male Balb/c mice to MBP by gavage at doses of 0, 25, 50, 100, 200 mg/kg for 14 days, and then evaluated the testicular alterations at the histological and molecular levels. MBP reduced mouse sperm count along with sperm malformation and seminiferous tubule degeneration in a dose-dependent manner. MBP dosed at 200 mg/kg significantly increased reactive oxygen species and malondialdehyde content in mouse testes. High doses of MBP (200 mg/kg) also significantly reduced mRNA expressions of testis growth and function related genes (Sox9 and Dazl). Our findings suggest that oxidative stress and down-regulated expression of Sox9 and Dazl may play important roles in MBP-induced testis injury.