Smart RNA Sequencing Reveals the Toxicological Effects of Diisobutyl Phthalate (DiBP) in Porcine Oocytes.

Diisobutyl phthalate (DiBP) is commonly used in the plastics industry, and recent studies have shown that environmental exposure and accumulation in the food chain caused inflammation in some organs. However, the underlying mechanisms by which DiBP affects oocyte quality have not yet been fully defined. We used immunostaining and fluorescence to evaluate the effects of DiBP exposure and demonstrated that it impaired the morphology of matured porcine oocytes through generation of cytoplasmic fragmentation, accompanied by the perturbed dynamics of the spindle and actin cytoskeleton, misdistributed endoplasmic reticulum, as well as partial exocytosis of cortical granules and ovastacin. Moreover, analysis of Smart RNA-seq found that DiBP-induced aberrant oocyte maturation could be induced by abnormal mitochondrial function and apoptosis. Importantly, we discovered that supplementation with pyrroloquinoline quinone (PQQ) significantly attenuated the meiotic abnormalities induced by DiBP exposure through the modulation of reactive oxygen species levels. Our findings demonstrated that DiBP exposure adversely affects oocyte meiotic maturation and that PQQ supplementation was an effective strategy to protect oocyte quality against DiBP exposure.

Extreme environmental doses of diisobutyl phthalate exposure induce oxidative stress and DNA damage in earthworms (Eisenia fetida): Evidence at the biochemical and molecular levels.

Diisobutyl phthalate (DIBP), as a plasticizer, is widely used and has caused many extreme soil contamination scenarios, posing potential risks to soil fauna. However, the toxic effects and mechanisms of DIBP on soil fauna remain unclear. In this study, earthworms (Eisenia fetida) were used as model animals to explore the subchronic toxicity of extreme DIBP soil exposure (300, 600, and 1200 mg/kg) for 28 days. The results showed that the level of reactive oxygen species (ROS) and the contents of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in E. fetida were significantly increased during continuous DIBP exposure. In addition, the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were significantly inhibited while glutathione S-transferase (GST) activity was activated during continuous exposure. Integrated biological response (IBR) analysis showed that DIBP had positive dose-dependent toxicity and negative time-dependent toxicity to E. fetida, and SOD/CAT were selected as sensitive biomarkers. The molecular docking study found that DIBP could stably bind to SOD/CAT through hydrogen bonding, which further proved its sensitivity. This study provides primary data for ecological and environmental risk assessment of extreme dose DIBP soil pollution.

Eucalyptol relieves the toxicity of diisobutyl phthalate in Ctenopharyngodon idellus kidney cells through Keap1/Nrf2/HO-1 pathway: Apoptosis-autophagy crosstalk and immunoregulation.

Diisobutyl phthalate (DiBP), one of the commonly used plasticizers in industry, is an endocrine disruptor and environmental contaminant that can persist in water and threaten the health of aquatic creatures. Eucalyptol (Euc), a monoterpenoid extracted from plants, has been proved to have anti-inflammatory, antioxidant, and detoxification properties. However, the protective mechanism of Euc against cell injury caused by DiBP exposure and the involvement of apoptosis, autophagy, and immunity remains unknown. In the current investigation, 27.8 µg/mL DiBP or/and 20 µM Euc has been applied to Ctenopharyngodon idellus kidney (CIK) cells for 24 h. The findings showed that exposure to DiBP raised intracellular ROS levels, inducing oxidative stress, and enhanced the rate of apoptosis as well as the expression of the apoptotic markers Bax, Caspase3, Caspase9, and Cytc while decreasing the expression of Bcl-2. Furthermore, DiBP inhibited IL-2, IFN-γ, Hepcidin-1, and ß-defensin expression and elevated TNF-α, and IL-1ß levels, causing immune dysfunction. DiBP and Euc co-treatment significantly activated the Keap1/Nrf2/HO-1 pathway, restored antioxidant enzyme activity, and elevated autophagy pathway-associated genes ATG5, Beclin1, and LC3B decreased p62 expression, enhanced cell autophagy, reduced apoptosis, and improved immunity. In conclusion, Euc promotes autophagy, alleviates DiBP-induced apoptosis, and improves immunological dysfunction in CIK cells by regulating the Keap1/Nrf2/HO-1 pathway. These results demonstrated the threat of DiBP exposure to fish while providing a theoretical foundation for using Euc in aquaculture.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli.

BACKGROUND: Phthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation. RESULTS: A carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min. CONCLUSIONS: This work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

Comparing the removal efficiency of diisobutyl phthalate by Bacillariophyta, Cyanophyta and Chlorophyta.

The utilization of microalgae for both removing phthalate esters (PAEs) from wastewater and producing bioenergy has become a popular research topic. However, there is a lack of studies comparing the effectiveness of different types of microalgae in removing these harmful compounds. Therefore, the present study aimed to evaluate and compare the efficiency of various processes, such as hydrolysis, photolysis, adsorption, and biodegradation, in removing diisobutyl phthalate (DiBP) using six different species of microalgae. The study indicated that the average removal efficiency of DiBP (initial concentrations of 5, 0.5, and 0.05 mg L-1) by all six microalgae (initial cell density of 1 × 106 cells mL-1) was in the order of Scenedesmus obliquus (95.39 %) > Chlorella vulgaris (94.78 %) > Chroococcus sp. (91.16 %) > Cyclotella sp. (89.32 %) > Nitzschia sp. (88.38 %) > Nostoc sp. (84.33 %). The results of both hydrolysis and photolysis experiments revealed that the removal of DiBP had minimal impact, with respective removal efficiencies of only 0.89 % and 1.82 %. The adsorption efficiency of all six microalgae decreased significantly with increasing initial DiBP concentrations, while the biodegradation efficiency was elevated. Chlorella vulgaris and Chroococcus sp. demonstrated the highest adsorption and biodegradation efficiencies among the microalgae tested. Scenedesmus obliquus was chosen for the analysis of the degradation products of DiBP due to its exceptional ability to remove DiBP. The analysis yielded valuable results, identifying monoisobutyl phthalate (MiBP), phthalic acid (PA), and salicylic acid (SA) as the possible degradation products of DiBP. The possible degradation pathways mainly included dealkylation, the addition of hydroxyl groups, and decarboxylation. This study lays a theoretical foundation for the elimination of PAEs in the aquatic environment.

Identification and characterisation of moderately thermostable diisobutyl phthalate degrading esterase from a Great Artesian Basin Bacillus velezensis NP05.

Phthalate esters are known to be endocrine disrupting chemicals and are documented to pollute environments. Enzymatic degradation of PAEs is a potential bioremedial strategy to manage contamination. Thermostable bioremedial enzymes have advantages in enzyme manufacturing and storage. In this study, we identified, overexpressed, and characterised a moderately thermostable para-nitrobenzyl esterase from whole genome sequencing of a Bacillus velezensis NP05 from the Great Artesian Basin, capable of sequential 2-step hydrolysis of diisobutyl phthalate. The pnbA enzyme has a molecular weight of 55.14 kDa and pI of 5.31. It preferentially degrades para-nitrophenyl butanoate and has an optimal pH of 7-8. The pnbA esterase has an optimal temperature of 55 °C with a half-life of 4 h. Using HPLC we found that pnbA (0.122 U) can hydrolyse 0.83 mM of DIBP within 25 min. Lastly, pnbA is potentially a more economically viable candidate for enzymatic bioremediation of diisobutyl phthalate as a free enzyme.

Developmental toxicity and mechanism of dibutyl phthalate and alternative diisobutyl phthalate in the early life stages of zebrafish (Danio rerio).

Diisobutyl phthalate (DiBP), is widely chemical replacement for Dibutyl phthalate (DBP). Although DBP and DiBP have been detected in surface water worldwide, few studies to date have systematically assessed the risks of DBP and its alternatives to aquatic organisms. The present study compared DBP and DiBP for their individual and joint toxicity as well as thyroid hormone levels in zebrafish embryo. Transcripts of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were investigated in developing zebrafish larvae by application of real time polymerase chain reaction. The median half-lethal concentrations of DBP and DiBP to zebrafish at 96 h were 0.545 mg L-1 and 1.149 mg L-1, respectively. The joint toxic effect of DBP-DiBP (0.25-0.53 mg L-1) with the same ratio showed a synergistic effect. Thyroid hormones levels increased with exposure to 10 µg L-1 of DBP or 50 µg L-1 of DiBP, and exposure to both compounds significantly increased thyroid gland-specific transcription of thyroglobulin gene (tg), hyronine deiodinase (dio2), and transthyretin (ttr), indicating an adverse effect associated with the HPT axis. Molecular docking results indicated that DBP (-7.10 kcal/M and -7.53 kcal/M) and DiBP (-6.63 kcal/M and -7.42 kcal/M) had the same docking energy with thyroid hormone receptors. Our data facilities an understand of potential harmful effects of DBP and its alternative (DiBP).

Diisobutyl phthalate (DiBP)-induced male germ cell toxicity and its alleviation approach.

Diisobutyl phthalate (DiBP) is a commonly used plasticizer in manufacturing consumer and industrial products to improve flexibility and durability. Despite of the numerous studies, however, the direct mechanism underlying the male reproductive damage of DiBP is poorly understood. In this study, we investigated the male germ cell toxicity of DiBP using GC-1 spermatogonia (spg) cells. Our results indicated that DiBP exposure causes oxidative stress and apoptosis in GC-1 spg cells. In addition, DiBP-derived autophagy activation and down-regulation of phosphoinositide 3-kinase (PI3K)-AKT and extracellular signal-regulated kinase (ERK) pathways further inhibited GC-1 spg cell proliferation, indicating that DiBP can instigate male germ cell toxicity by targeting several pathways. Importantly, a combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine significantly reduced DiBP-induced male germ cell toxicity and restored proliferation. Taken together, the results of this study can provide valuable information to the existing literature by enhancing the understanding of single phthalate DiBP-derived male germ cell toxicity and the therapeutic interventions that can mitigate DiBP damage.

Development of ecological risk assessment for Diisobutyl phthalate and di-n-octyl phthalate in surface water of China based on species sensitivity distribution model.

Phthalic acid esters (PAEs) are commonly used as plasticizer and are emerging concern worldwide for potent adverse effects of aquatic organisms. Certain PAEs were often detected in different environmental matrices but related toxicity data were still lacking to support their risk assessment. The study investigated the acute toxicity of Diisobutyl phthalate (DiBP) and Di-n-octyl phthalate (DnOP) using 6 Chinese resident aquatic organisms from 3 phyla and 6 species and constructed the species sensitivity distribution (SSD) models for ecological risk assessment. Lethal concentration 50% (LC50) ranges of DiBP and DnOP were 4.89-21.45 mg/L and 1.45-1200 mg/L, respectively. The derived acute and chronic predicted no-effect concentrations (PNECs) based on log-normal model of water were 0.54 and 0.04 mg/L for DiBP and 0.23 and 0.05 mg/L for DnOP, respectively. The ERA for DiBP and DnOP in the surface water and sediment of China was conducted. Water samples of Haihe Rive (RQ = 0.41) and Hun River (RQ = 0.16) of DiBP showed medium risk. And sediment samples of Yellow River (RQ = 0.71) and Chao Hu Lake (RQ = 0.42) of DiBP showed medium risk. Meanwhile, the above water and sediment samples (RQ＜0.1) of DnOP showed low risk.

Toxicokinetics of diisobutyl phthalate and its major metabolite, monoisobutyl phthalate, in rats: UPLC-ESI-MS/MS method development for the simultaneous determination of diisobutyl phthalate and its major metabolite, monoisobutyl phthalate, in rat plasma, urine, feces, and 11 various tissues collected from a toxicokinetic study.

The aim of this study was to explore the toxicokinetics of diisobutyl-phthalate (DiBP) and its major metabolite, monoisobutyl-phthalate (MiBP), by developing a UPLC-ESI-MS/MS method for simultaneously measuring DiBP and MiBP in rat plasma, urine, feces, and 11 different tissues. For the experiment, 0.1% (v/v) aqueous formic acid and acetonitrile mobile phase by gradient elution at a flow rate of 0.3 mL/min, equipped with a KINETEX core-shell C18-column (50 × 2.1 mm, 1.7 µm), was used to completely separate analytes. The mass transitions were m/z 279.1 â†’ 149.0 for DiBP, 221.0 â†’ 77.0 for MiBP, and 283.2 â†’ 153.0 for DiBP-d4 as an internal standard. The developed assay had lower limits of quantification of 0.01 ng/mL for DiBP and 0.1 ng/mL for MiBP at all biological matrices. Toxicokinetics of DiBP were characterized by extensive distribution, short half-life, and high clearance. DiBP was rapidly metabolized to MiBP, with MiBP levels consistently exceeding the DiBP levels. Distribution of MiBP to tissues was considerable. The developed analytical method satisfied international criteria and was successfully applied to toxicokinetic studies after oral and intravenous administration of DiBP to rats. Findings of this study may be useful for evaluating the external exposure and toxic potential of DiBP and its metabolite in risk assessment.

The reproductive toxicity and potential mechanisms of combined exposure to dibutyl phthalate and diisobutyl phthalate in male zebrafish (Danio rerio).

Dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) are phthalate compounds frequently detected in the environment. Despite increasing awareness of their toxicity in human and animals, the male reproductive toxicity of their combined exposure remains elusive. The purposes of this study were to investigate whether combined exposure to DBP and DiBP could induce male reproductive toxicity, and to explore the potential toxicological mechanisms. Adult male zebrafish were exposed to DBP (11, 113 and 1133 µg L-1), DiBP (10, 103 and 1038 µg L-1) and their mixtures (Mix) (11 + 10, 113 + 103, 1133 + 1038 µg L-1) for 30 days, and their effects on plasma hormone secretion, testis histology and transcriptomics were examined. Highest concentrations of Mix exposure caused greater imbalance ratio of T/E2 and more severe structural damage to testis than single exposure. These effects were consistent with the testis transcriptome analysis for which 4570 genes were differentially expressed in Mix exposure, while 2795 and 1613 genes were differentially expressed in DBP and DiBP, respectively. KEGG pathway analysis showed that both single and combined exposure of DBP and DiBP could affect cytokine-cytokine receptor interaction. The difference was that combined exposure could also affect steroid hormone synthesis, extracellular matrix receptor interaction, retinol metabolism, and PPAR signaling pathways. These results demonstrated that combined exposure to DBP and DiBP could disrupt spermatogenesis and elicit male reproductive toxicity in zebrafish.

Ordinal dose-response modeling approach for the phthalate syndrome.

BACKGROUND: The phthalate syndrome (PS) is a collection of related male reproductive developmental effects, ranging in severity, that have been observed in rats after gestational exposure to developmentally-toxic phthalates. For statistical purposes, the PS is defined as a single endpoint and one dose-response analysis is conducted, rather than conducting multiple analyses on each individual endpoint. OBJECTIVE: To improve dose-response modeling approaches for the PS and other syndromes of effects by accounting for differing severity levels among the endpoints. METHODS: Ordinal dose-response modeling was performed on PS data from a published study of diisobutyl phthalate (DIBP) gestational exposure to male Sprague-Dawley rats. To incorporate PS endpoint severity, the endpoints were categorized into ordinal levels based on the expected impact of male developmental endpoint's on fertility. Then, a benchmark dose was estimated for each ordinal level. A bootstrap procedure was used to account for the nested nature of the data, and a sensitivity analysis was performed to assess the bootstrap results. A comparison of the estimates between the ordinal and the dichotomous model was performed. RESULTS: The ordinal version of the log-logistic model applied to the data categorized by PS endpoint severity level provided benchmark dose estimates that were closer to each other in value and had lower variability than the traditional dichotomous application. The sensitivity analysis confirmed the validity of the bootstrap results. CONCLUSION: The ordinal dose-response modeling method accounts for severity differences among dichotomous PS endpoints, can be expanded in the future to include more severity levels, and can be used in both single and cumulative phthalate risk assessments.

Assessment of phthalate ester residues and distribution patterns in Baijiu raw materials and Baijiu.

Phthalate esters (PAEs) are harmful to human health and have been repeatedly identified in Baijiu samples. In our study, the distribution and degradation characteristics of 14 PAEs in Baijiu raw materials (BRMs) and Baijiu during distillation were detected using QuEChERS or vortex-assisted surfactant-enhanced-emulsification liquid-liquid micro-extraction (VSLLME) methods coupled with gas chromatography-mass spectrometry. The same five PAEs were detected in all tested samples, values ranged from 0.003 to 0.292 mg/kg; however, higher concentrations existed in BRMs compared to Baijiu samples. Using multivariate statistical analysis, detailed distinctions between different varieties of Baijiu and BRMs and separation-related PAE markers were revealed. PAEs concentration during Baijiu distillation showed a decreasing trend. The highest concentrations detected in distillate heads, were 1.6-, 2.3-, and 8.1-fold higher than those in heart1, heart2, and tail distillates, respectively. These findings revealed that PAEs may migrate from BRMs; moreover, that PAEs content can be regulated by distillation.

Removal of phthalic acid esters from sea buckthorn (Hippophae rhamnoides L.) pulp oil by steam distillation and molecular distillation.

Sea buckthorn (Hippophae rhamnoides L.) pulp oils (SPOs) are rich in a variety of beneficial bioactive ingredients. Nevertheless, SPOs would be exposed to plastic equipment during processing, resulted in increasing phthalates contents and edible risk, as well as affecting oil quality. For these reasons, the effects of two stages steam distillation (SD2) and two stages molecular distillation (MD2) on phthalic acid esters (PAEs) content were investigated and compared in the present work. Compared with SD2, MD2 showed higher removal rates of seven selected PAEs from the SPO. Even if the initial concentration of DBP and DEHP in R-SPO were 1.626 and 10.933 mg/kg respectively, the concentration of DBP and DINP could be reduced below the limit set by China government after treated with MD2. Besides that, there was no trans-fatty acids generated in SPO during the distillation process.

Solid phase microextraction of phthalic acid esters from vegetable oils using iron (III)-based metal-organic framework/graphene oxide coating.

A hybrid composite featuring an iron-based metal-organic framework Material of Institute Lavoisier-88(Fe) and graphene oxide (MIL-88(Fe)/GO) was synthesized and used as the solid-phase microextraction (SPME) coating. The SPME fiber was prepared by covalent bonding of the MIL-88(Fe)/GO composite onto the stainless steel substrate. The fiber had a good durability and allowed >100 replicate extractions. The developed method, which combined the MIL-88(Fe)/GO coated fiber based SPME with gas chromatography-flame ionization detection (GC-FID), achieved low limits of detection (0.5-2.0 ng g-1, S/N = 3) and good linearity (r2 > 0.994) for the phthalic acid esters (PAEs) from various vegetable oil samples. The repeatability and fiber-to-fiber reproducibility were in the range of 4.0-9.1% and 5.7-11.4%, respectively. The method was successfully applied to the analysis of PAEs from vegetable oil samples with good recoveries (83.1-104.1%) and satisfactory precisions (RSDs < 10.5%), indicating that the MIL-88(Fe)/GO hybrid composite is a good coating material for the SPME of PAEs.

A review of human biomonitoring data used in regulatory risk assessment under Canada's Chemicals Management Program.

As a part of the Chemicals Management Plan launched in 2006, the Government of Canada is assessing and managing, where appropriate, the potential health and ecological risks associated with approximately 4300 substances under the Canadian Environmental Protection Act (1999). Since that time, nearly 3000 substances have been assessed, with human biomonitoring (HBM) data playing an increasingly important role for some substances. Case studies are presented, including both inorganic and organic substances (i.e., selenium, triclosan, phthalates), which highlight the impact and overall role HBM has had in regulatory decision making in Canada for these three substances as well as criteria used in the application of HBM data in human health risk assessment. An overview of its limitations in terms of how and when HBM data can be applied, when assessing human health in a regulatory setting, is discussed as well as the role HBM data can play in priority setting.

Multiresidue analysis of environmental pollutants in edible vegetable oils by gas chromatography-tandem mass spectrometry.

A novel multiresidue determination of polycyclic aromatic hydrocarbons (PAHs), phthalate esters (PAEs) and alkylphenols (APs) in edible vegetable oils was developed. The samples were extracted with hexane-saturated acetonitrile, and after concentration, the extract was directly qualitatively and quantitatively analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS) with multiple reaction monitoring (MRM) in positive ion mode. The calibration curve displayed good linearity in the range of 2-100 µg/L, with correlation coefficients greater than 0.99. The mean recoveries were 70.0-110.8% by analysis of spiked oil, and the relative standard deviations (RSDs) were 2.1-10.2% (n=6), respectively. The limits of detection (LODs) for the 23 PAHs, 17 PAEs and 3 APs were 0.1-1.0 µg/kg, 0.1-4.0 µg/kg and 1.2-3.0 µg/kg, respectively. The established method effectively avoided interference from large amounts of lipids and pigments. It was applied to real sample and shown to be a rapid and reliable alternative for determination and confirmation in routine analysis.

Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples.

The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate-bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R(2) = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography-mass spectrometry (GC-MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples.

Exposure to bisphenol A, but not phthalates, increases spontaneous diabetes type 1 development in NOD mice.

Type 1 diabetes mellitus (T1DM) is an autoimmune destruction of insulin producing pancreatic beta-cells due to a genetic predisposition and can be triggered by environmental factors. We have previously shown that bisphenol A (BPA) accelerates the spontaneous development of diabetes in non-obese diabetic (NOD) mice. Here, we hypothesized that oral exposure to a mixture of the endocrine disruptors BPA and phthalates, relevant for human exposure, would accelerate diabetes development compared to BPA alone. NOD mice were exposed to BPA (1 mg/l), a mixture of phthalates (DEHP 1 mg/l, DBP 0.2 mg/l, BBP 10 mg/l and DiBP 20 mg/l) or a combination of BPA and the phthalate mixture through drinking water from conception and throughout life. Previous observations that BPA exposure increased the prevalence of diabetes and insulitis and decreased the number of tissue resident macrophages in pancreas were confirmed, and extended by demonstrating that BPA exposure also impaired the phagocytic activity of peritoneal macrophages. None of these effects were observed after phthalate exposure alone. The phthalate exposure in combination with BPA seemed to dampen the BPA effects on macrophage number and function as well as diabetes development, but not insulitis development. Exposure to BPA alone or in combination with phthalates decreased cytokine release (TNFα, IL-6, IL-10, IFNγ, IL-4) from in vitro stimulated splenocytes and lymph node cells, indicating systemic changes in immune function. In conclusion, exposure to BPA, but not to phthalates or mixed exposure to BPA and phthalates, accelerated diabetes development in NOD mice, apparently in part via systemic immune alterations including decreased macrophage function.

Chemical constituents from roots of Eupatorium chinense / 中草药

Objective To study chemical constituents of the roots of Eupatorium chinense. Methods The chemical constituents were separated and purified by the normal phase silica gel column chromatography, preparative thin-layer chromatography, and preparative HPLC. Their structures were determined by various spectral data. Results Nineteen compounds were isolated from the acetic ether extract of E. chinense and the structures were identified as euparin (1), 1-[2-(1-acetoxymethyl-vinyl)-6-hydroxy-benzofuran-5-yl]- ethanone (2), 6-hydroxy-3β-methoxytrematone (3), euparone (4), 8-methoxy-9-hydroxythymol (5), dehydroespeleton (6), 8-methoxy-9-hydroxythymol 3-O-angelate (7), 9-hydroxythymol (8), 4-hydroxy cinnamic acid methyl ester (9), p-hydroxy benzaldehyde (10), 8,9-dehydro-10-hydroxythymol (11), diisobutyl phthalate (12), dibutyl phthalate (13), p-coumaric acid (14), dihydrocoumarin (15), methylcaffeate (16), 2,5-dimethylphenol (17), 1H-indazole (18), and (Z)-3-(hydroxymethyl)-7-methylocta- 2,6-dienly1 acetate (19). Conclusion The chemical constituents are investigated and identified from the roots of E. chinense for the first time. Among them, compounds 2, 3, 5-11 are isolated from E. chinense for the first time, and compounds 6, 9, 12, 14-19 are isolated from the genus of Eupatorium for the first time. Compounds 1-4 are benzofurans, which are the characteristic constituents in Eupatorium genus.