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Mutations in leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GBA) represent two most common genetic causes of Parkinson’s disease (PD). Both genes are important in the autophagic-lysosomal pathway (ALP), defects of which are associated with α-synuclein (α-syn) accumulation. LRRK2 regulates macroautophagy via activation of the mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) and the calcium-dependent adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathways. Phosphorylation of Rab GTPases by LRRK2 regulates lysosomal homeostasis and endosomal trafficking. Mutant LRRK2 impairs chaperone-mediated autophagy, resulting in α-syn binding and oligomerization on lysosomal membranes. Mutations in GBA reduce glucocerebrosidase (GCase) activity, leading to glucosylceramide accumulation, α-syn aggregation and broad autophagic abnormalities. LRRK2 and GBA influence each other: GCase activity is reduced in LRRK2 mutant cells, and LRRK2 kinase inhibition can alter GCase activity in GBA mutant cells. Clinically, LRRK2 G2019S mutation seems to modify the effects of GBA mutation, resulting in milder symptoms than those resulting from GBA mutation alone. However, dual mutation carriers have an increased risk of PD and earlier age of onset compared with single mutation carriers, suggesting an additive deleterious effect on the initiation of PD pathogenic processes. Crosstalk between LRRK2 and GBA in PD exists, but its exact mechanism is unclear. Drugs that inhibit LRRK2 kinase or activate GCase are showing efficacy in pre-clinical models. Since LRRK2 kinase and GCase activities are also altered in idiopathic PD (iPD), it remains to be seen if these drugs will be useful in disease modification of iPD.

Salidroside (Sal), the major active constituent of

Objective This study investigated the correlations of digit ratio (relative length of second and fourth fingers, 2D: 4D) and muscular strength with the progression of chronic obstructive pulmonary disease (COPD). Methods In total, 164 patients with COPD were enrolled in this prospective study. In all patients, the following parameters were measured: body compartments, pulmonary function, digit ratio (i.e., 2D: 4D), muscle function, and levels of partial pressure of oxygen and partial pressure of carbon dioxide in arterial blood. Results The right-hand digit ratio (R2D: 4D) was associated with dominant hand muscle dysfunction, non-dominant hand muscle dysfunction, and inspiratory muscle dysfunction. Logistic regression analysis showed that right-hand 2D: 4D (odds ratio [ OR ] = 0.01) and dominant hand muscle dysfunction ( OR = 5.60) were significantly associated with past hospital admission. After adjustment for forced expiratory volume in 1 second, the following factors were associated with present acute exacerbations: right-hand 2D: 4D ( OR = 0.01), dominant hand muscle dysfunction ( OR = 3.83), expiratory muscle dysfunction ( OR = 3.80), and inspiratory muscle dysfunction ( OR = 3.61). Conclusions Lower digit ratio may be associated with higher prevalence of muscle dysfunction. This factor could be used to identify patients with COPD who are at higher risk of acute exacerbation.

Noroviruses are the leading cause of acute gastroenteritis and food-borne diseases worldwide. Thus, a rapid, accurate, and easy-to-implement detection method for controlling infection and monitoring progression is urgently needed. In this study, we constructed a novel sandwich-type electrochemical biosensor integrated with two specific recognition elements (aptamer and peptide) for human norovirus (HuNoV). The electrochemical biosensor was fabricated using magnetic covalent organic framework/pillararene heterosupramolecular nanocomposites (MB@Apt@WP5A@Au@COF@Fe3O4) as the signal probes. The sensor showed high accuracy and selectivity. The detection method does not need the extraction and amplification of virus nucleic acid and has a short turn-around time. Intriguingly, the proposed biosensor had a limit of detection of 0.84 copy mL-1 for HuNoV, which was the highest sensitivity among published assays. The proposed biosensor showed higher sensitivity and accuracy compared with immunochromatographic assay in the detection of 98 clinical specimens. The biosensor was capable of determining the predominant infection strain of GII.4 and also GII.3 and achieved 74% selectivity for HuNoV GII group. This study provides a potential method for point-of-care testing and highlights the integrated utilization of Apt and peptide in sensor construction.

The calcium phosphate [Ca3(PO4)2] precipitation was used for improving the clarification efficiency in harvest process of the monoclonal antibody (mAb) containing cell culture fluid (CCF) with high turbidity and product titer. The flocculation conditions (concentration, addition order of flocculants, pH, and operation time), and the effect of flocculants on the mAb physical chemical properties (such as distribution of charge variants and aggregates) and process-related impurities removal (such as DNA and CHOP) were evaluated in this study. The results showed that the turbidity of CCF supernatant was significantly reduced at pH 7, 120 min with addition of phosphate ions first, while a high mAb recovery yield was kept in the CCF supernatant after flocculation. Addition of calcium ions at 15-60 mM was sufficient for flocculation in this study. A relationship between turbidity/mAb recovery yield and the concentration of calcium ions was established. More than 85% DNA in the CCF were effectively removed by the addition of optimal concentration of flocculants. Flocculation process of Ca3(PO4)2 is an effective pretreatment method in purification processes of mAbs from the CCF with high turbidity and product titer.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC). Patients with intermediate and advanced stage NPC receiving only radiotherapy have limited survival, so newer immunotherapeutic approaches are sought. The major impediment to better clinical outcomes is tumor immune tolerance. Indoleamine 2,3-dioxygenase (IDO), an IFNγ-inducible enzyme, is a major inducer of immune tolerance during tumor development; therefore, inhibition of the IDO pathway is an important modality for cancer treatment. We show that bortezomib, a proteasomal inhibitor, inhibited the pathways leading to STAT1 and IRF-1 activation, both of which are necessary for IDO expression. Bortezomib downregulated IFNγ-induced IDO expression via inhibition of STAT1 phosphorylation and nuclear translocation, thereby suppressing STAT1-driven IDO transcription in NPC cells. Bortezomib also promoted IκB-α phosphorylation-ubiquitination, which released NF-κB from IκB-α. However, the released NF-κB could not enter the nucleus to conduct its biological effects and accumulated in the cytoplasm. Negative feedback inhibited the transcription of NF-κB, which is important for activating IRF-1 expression. IDO expression is regulated by two important transcription factor binding sites, ISREs, which bind STAT1 and IRF-1, and GASs, which binds STAT1. Bortezomib upregulated IRF-1 protein by inhibiting its proteasome-dependent degradation, but it also inhibited STAT1 phosphorylation, which directly inhibited the activation of GAS and indirectly inhibited the activation of ISRE, which needs both STAT1 and IRF-1. These discoveries provide a mechanism for the antitumor action of bortezomib and have implications for the development of clinical cancer immunotherapy for preventing and treating NPC. Cancer Immunol Res; 5(1); 42–51. ©2016 AACR.

To investigate the effect of ILC2s on Th2-type adaptive immunity during the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), the study enrolled healthy people, stable COPD patients, and AECOPD patients. Flow cytometry was used to detect Th1, Th2, and ILC2 in the peripheral blood and CD80 and MHC II levels on ILC2. The mRNA levels of GATA3, RORα, and CRTH2 of ILC2s were detected by RT-PCR. In addition, ILC2s from the peripheral blood of AECOPD patients were cocultured with CD4+ T cells from the peripheral blood of healthy controls. Cytokine levels in serum of the three groups and the in vitro coculture supernatants were measured by ELISA. Compared with the stable COPD group or the healthy control group, Th2 in the peripheral blood of AECOPD group increased dramatically, inducing an increase of Th2/Th1 ratio in AECOPD patients. Meanwhile, the level of IL-4 in the serum of this group was also increased. However, we also detected ILC2s in the peripheral blood of the AECOPD group and found that it was also increased, alone with the increased GATA3, RORα, and CRTH2 mRNA levels. We also found that the CD80 and MHC II on ILC2 were significantly upregulated and the proportion of MHC II+ ILC2 cells was significantly positively correlated with the proportion of Th2 cells in AECOPD patients. To further demonstrate the effect of ILC2 on Th2 cells, we cocultured ILC2 with CD4+ T cells in vitro, which also showed a significant increase of Th2 ratio as well as Th2-associated cytokines IL-4, IL-5, and IL-13. However, we found that this effect of ILC2s on Th2 cells could be inhibited by the addition of anti-MHC II. The Th2/Th1 balance shifts to Th2 in AECOPD. ILC2s may function as APC by the upregulation of MHC II and regulate adaptive immunity shift to Th2-type response in AECOPD.

// Guan-Min Jiang 1,* , Wei Xu 2,* , Jun Du 3 , Kun-Shui Zhang 4 , Qiu-Gui Zhang 2 , Xiao-Wei Wang 1 , Zhi-Gang Liu 5 , Shuang-Quan Liu 2 , Wan-Ying Xie 2 , Hui-Fang Liu 2 , Jing-Shi Liu 6 and Bai-Ping Wu 1 1 Department of Clinical Laboratory, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China 2 Department of Clinical Laboratory, The First Affiliated Hospital of University of South China, Hengyang, Hunan, China 3 Department of Microbial and Biochemical Pharmacy School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 4 Department of Pharmacy, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China 5 Department of Radiation Oncology, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China 6 Department of Anesthesia, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China * These authors have contributed equally to this work Correspondence to: Guan-Min Jiang, email: // Bai-Ping Wu, email: // Jing-Shi Liu, email: // Keywords : fibroblast activation protein α, tumor microenvironment, immune suppression, immunotherapy Received : December 04, 2015 Accepted : February 28, 2016 Published : March 15, 2016 Abstract Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells (stromal and cancerous) are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target.

In order to identify biomarkers for early diagnosis and/or for therapeutic targets in the delayed health effects of ionizing radiation, we analyzed the subgroups of lymphocytes, serum protein levels and gene expression profiles in the peripheral blood of three 60Co γ-ray accidentally exposed persons during the three years after irradiation. Flow cytometry analyses and agarose gel electrophoresis were applied to investigate the subgroups of lymphocytes and the composition of serum proteins, respectively. Gene expression profiling was obtained using a whole genome gene expression chip assay. Both the percentage of CD4+ T lymphocytes and the ratio of Th to Ts were reduced compared with the normal control values. The percentage of albumin decreased whereas beta globulin increased. There were 285 up-regulated and 446 down-regulated genes in irradiated samples relative to the control samples. The expression of KDR, CEACAM8 and OSM was validated by RT-PCR. The majority of the differentially expressed genes encode proteins associated with the immune response, inflammation, oncogenesis, cell structure, oxidative stress, neuro-hormone regulation, reproduction, susceptibility to psychiatric disorders, or transcriptional regulation. We have identified a number of promising novel candidates that have potential for serving as biomarkers for delayed damage. Furthermore, the changes in the immunological indicator CD4+ T cells, and the ratio of CD4+ T to CD8+ T cells may be biomarkers for the prediction of delayed damage by ionizing radiation. The findings of our study are useful for forming a comprehensive understanding of the mechanisms underlying the delayed effects of ionizing radiation.

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal survival in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential (MMP). NF-κB regulates neuronal viability via its complexes, p65 mediating cell death and c-Rel promoting cell survival. We reported previously that NF-κB mediates UCP4 neuroprotection against MPP(+) toxicity. Here, we investigated its link with the NF-κB c-Rel prosurvival pathway in alleviating mitochondrial dysfunction and oxidative stress. We overexpressed a c-Rel-encoding plasmid in SH-SY5Y cells and showed that c-Rel overexpression induced NF-κB activity without affecting p65 level. Overexpression of c-Rel increased UCP4 promoter activity and protein expression. Electrophoretic mobility shift assay showed that H(2)O(2) increased NF-κB binding to the UCP4 promoter and that NF-κB complexes were composed of p50/p50 and p50/c-Rel dimers. Under H(2)O(2)-induced oxidative stress, UCP4 knockdown significantly increased superoxide levels, decreased reduced glutathione (GSH) levels, and increased oxidized glutathione levels, compared to controls. UCP4 expression induced by c-Rel overexpression significantly decreased superoxide levels and preserved GSH levels and MMP under similar stress. These protective effects of c-Rel overexpression in H(2)O(2)-induced oxidative stress were significantly reduced after UCP4 knockdown, indicating that UCP4 is a target effector gene of the NF-κB c-Rel prosurvival pathway to mitigate the effects of oxidative stress.

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP+ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP+-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP+ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP+-induced toxicity., published_or_final_version, Springer Open Choice, 01 Dec 2010

Background Electroacupuncture (EA) treatment has been shown to increase bone mineral density (BMD) in ovariectomised (OVX) rats; however, the underlying mechanisms remain unclear. Objective To systematically evaluate the effects of EA on OVX rats and the Wnt/β-catenin signalling pathway. Methods Three-month-old female Sprague–Dawley rats were randomly divided into three different groups (n=10 each): sham operated control (sham operated), ovariectomy (OVX) and ovariectomy with EA treatment (OVX+EA). Rats in the OVX+EA group received 12-week EA treatments. Results Serum bone-specific alkaline phosphatase level (pConclusion This study demonstrates that EA can prevent OVX-induced bone loss and deterioration of bone architecture and strength by stimulating the Wnt/β-catenin signalling pathway. These findings suggest that EA may bet a promising adjunct method for inhibiting OVX-induced osteoporosis in clinical settings.

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP +), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p, published_or_final_version

To investigate the prevalence and percentage of dyslipidemia awareness among overweight and obese residents in Beijing community.Cross-sectional data of 9786 subjects from capital cholesterol education and intervention program (CCEIP) were analyzed. Participants were divided into 3 groups (Normal, overweight and obese) based on body mass index (BMI). Blood lipid levels were determined from overnight fasting plasma samples. Questionnaires were collected to estimate awareness of dyslipidemia.(1) Blood lipids levels were positively correlated with BMI (r = 0.17, 0.18, -0.26 and 0.35 between total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride and BMI, respectively, all P0.01). (2) Prevalence of dyslipidemia increased in proportion with increase of BMI. The age-standardized prevalence of dyslipidemia of normal, overweight and obese population was 23. 9%, 43.3% and 65.4% in men and 17.9%, 29.2% and 42.3% in women. Dyslipidemia was more frequent in obese men than obese women (65.4% vs. 42.3%, P0.01). However, this gender difference decreased gradually with the increase of age. Risk of hypercholesterolemia, high LDL-C, low HDL-C and hypertriglyceridemia in obese men was 1.6, 2.9, 2.4, and 2.7 folders higher than in the normal body weight men and was 1.3, 1.9, 1.7 and 2.1 folders higher in obese women than in normal body weight women. (3) Unexpectedly, the percentage of dyslipidemia awareness in overweight and obese population was not significantly higher than in the normal body weight group (P0.05).The prevalence of dyslipidemia was high while the percentage of dyslipidemia awareness was rather low in obese population. Aggressive intervention should be taken in obese population, especially in the young obese men, to effectively reduce dyslipidemia.

We explored the protective mechanisms of human neuronal mitochondrial uncoupling protein-5 (UCP5) in MPP+- and dopamine-induced toxicity after its stable overexpression in SH-SY5Y cells. We raised specific polyclonal antibodies. Overexpressed UCP5 localized in mitochondria but not in cytosol. UCP5 overexpression increased proton leak, decreased mitochondrial membrane potential (MMP), reduced ATP production, and increased overall oxygen consumption (demonstrating uncoupling activity). UCP5 overexpression did not affect other neuronal UCP expression (UCP2 and UCP4). Overexpressing UCP5 is protective against MPP+- and dopamine-induced toxicity. MPP+ and dopamine exposure for 6 h reduced MMP and increased superoxide levels. ATP levels in UCP5-overexpressing cells were preserved under MPP+ and dopamine toxicity, comparable to levels in untreated vector controls. At 24 h, UCP5 overexpression preserved MMP, ATP levels, and cell survival; attenuated superoxide generation; and maintained oxidative phosphorylation as indicated by lower lactate levels. MPP+ and dopamine exposure induced UCP5 mRNA transcription but did not decrease transcript degradation, as inhibition of transcription by actinomycin-D abolished induction by either toxin. Compared with our previous studies on UCP4, we observed functional differences between UCP4 and UCP5 in enhancing mitochondrial efficiency. These neuronal UCP homologues may work synergistically to maintain oxidative balance (through uncoupling activities) and ATP production (by modifying MMP).

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal cell survival in MPP(+)-induced toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential. UCP4 expression is increased by MPP(+), but its regulation is unknown. Using serial human UCP4 promoter-luciferase reporter gene constructs, we identified and characterized several cis-acting elements that can regulate UCP4 expression. Core promoter activity exists within 100 bp upstream of the transcription initiation site (TIS=+1). Both CAAT box (-33/-27) and Sp1 (-62/-49) elements are crucial and act synergistically in its transcription. We identified a NF-kappaB putative binding site at -507/-495. Mutation of this site significantly decreased UCP4 promoter activity. Activation of NF-kappaB by TNFalpha or cycloheximide increased, whereas its inhibition by 4-hydroxy-2-nonenal or transfection of pIkappaBalphaM suppressed, UCP4 promoter activity. NF-kappaB inhibition significantly suppressed the MPP(+)-induced increase in UCP4 expression. MPP(+) increased specific binding of NF-kappaB protein complexes to this site in electrophoretic mobility shift assay. Both UCP4 knockdown and NF-kappaB inhibition exacerbated MPP(+)-induced cell death. We present the first direct evidence that UCP4 is regulated by NF-kappaB, mediated via a functional NF-kappaB site in its promoter region, and that UCP4 has a significant role in NF-kappaB prosurvival signaling, mediating its protection against MPP(+) toxicity.

To identify the gene mutation type of an inherited coagulation factor XIII (FXIII) deficiency pedigree.PCR and DNA sequencing were used to identify the mutations in the 15 exons and the flank sequence of FXIII gene in the proband. The identified mutations were validated by allele specific PCR, PCR restriction fragment length polymorphism technique or DNA sequencing in the family members and 100 healthy volunteers.Arg77Cys and Argl74stop double heterozygous mutations were discovered in the proband. The pedigree analysis showed that Arg77Cys missense mutation inherited from her father, and Arg174stop from her mother. The Arg77Cys missense mutation in exon 3 was not found in her husband and the other 100 healthy volunteers.A novel Arg174stop nonsense mutation was discovered in human FXIII gene. A simple DNA assay based on PCR for detection of this mutation was developed. The congenital FXIII deficiency in the proband might be caused by the coinheritance of the Arg77Cys missense mutation in exon 3 and the Arg174stop nonsense mutation in exon 4.

To discover the mutations of human blood coagulation factor V (FV) gene in a Chinese family with congenital factor V deficiency, and to explore the molecular mechanism associated with the congenital factor V deficiency.PCR and DNA sequencing were used to look for the FV gene mutations in the proband. And the novel mutation were testified by PCR restriction fragment length polymorphism technique or reverse DNA sequencing. One hundred healthy volunteers were chosen as controls at random.Two novel mutations were discovered in the FV gene of proband, which were the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16 (G--T). The pedigree analysis showed that the two mutations inherited from his parents respectively: the A1763C came from his father, and the G--T from his mother. The A1763C missense mutation in exon 11 was not found in each of 100 healthy volunteers.The congenital deficiency of FV in the proband might be caused by the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16, which jointly caused the proband to be a double heterozygote.

To investigate the clinical manifestations, complications and treatment of medicament-like dermatitis due to trichloroethylene (TCE), so as to provide basis for studying its etiology and mechanism.Fifty patients with dermatitis due to TCE from 1997 to 2000 were analysed retrospectively.The occurrence of the dermatitis was not parallel to TCE exposure levels, without significant dose-effect relationship. This disease could be caused by both inhalation and skin exposure. The latency period of TCE dermatitis ranged from 5 to 66 days, and the average was 31.5 d (Medium). The major clinical manifestations included skin lesions, fever, superficial lymph node swelling and liver dysfunction. Infection was the major complication. Glucocorticoid was effective for treatment of this disease.The clinical manifestations due to TCE exposure were similar to dermatitis medicamentosa. The major clinical types of TCE dermatitis included exfoliative dermatitis and erythema multiforme. The dermatitis is considered to be mediated by delayed-type (IV) hypersensitivity. The key factors to treat this disease successfully included the use of glucocorticoid in time with sufficient dose and full course, professional skin care, active treatment to protect the liver and to avoid infection.

To study the dioxin level of breast milk among Chinese mothers, and to assess the dioxin intake of new-born babies from mother's milk and compare with the Tolerable Daily Intake (TDI) of dioxin.The CALUX bioassay was used to detect the dioxin concentration of the first time mother's milk among the inland samples (Shenyang region; 32 cases) and the coastal city samples (Dalian region; 47 cases).The median value of the dioxin Toxic Equivalence (TEQ) in breast milk in the Dalian region was 15.84 pg TEQs.g(-1) fat, which was significantly higher than that in the Shenyang region 7.21 pg TEQs.g(-1) fat (P0.01).The dioxin level in breast milk in Chinese is at the world's average level. The dioxin intake of the new-born babies during the period of lactation was higher than the lowest limit of the Tolerable Daily Intake (TDI) proposed by WHO. This situation should be noticed by the related authorities.

Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p

1. Glucuronidation of clofibric acid, the pharmacologically active form of the hypolipidemic drug clofibrate was investigated in a human population, either in vitro with liver homogenates from biopsies, or after ingestion of the drug and determination of the urinary metabolite. No difference in the glucuronidation rate according to age of the patients was observed. Bilirubin but not clofibric acid glucuronidation was significantly higher in women (106% increase), when expressed per gram of tissue. 2. The excretion of clofibryl glucuronide in women who took oral contraceptives was significantly enhanced by 25%. 3. In female rats, treatment with the contraceptive agent norethindrone also stimulated by 48% the formation of clofibrylglucuronide in liver microsomes.The effect of oral contraceptive (OC) intake on the glucuronidation of clofibric acid was investigated both in vivo, by measuring the excretion of urinary clofibrylglucuronide in female Wistar rats, or in vitro, with liver biopsy homogenates from 69 health female volunteers. In both study groups norethindrone markedly enhanced glucuronidation of clofibric acid while the situation was modulated by the administration of ethinyl estradiol. Excretion of clofibric acid into urine averaged 142.1 + or - 38.4 mg in OC users compared to only 105.8 + or - 31.5 mg in non-users. When expressed per gram of tissue, bilirubin but not clofibric acid glucuronidation was significantly greater in human liver biopsies from OC users compared to non-users. In rats treated with norethindrone, the liver microsomal protein content was increased 28% over the average control value and formation of clofibylglucoride was stimulated by 48%. It is hypothesized that the stimulation of clofibylglucuronide in OC users results from the selective stimulation in the liver of the isoenzyme involved in the process of the glucuronidation of clofibric acid.

An ion-pair high-performance liquid chromatographic technique has been developed to reduce the elution times of both glucuronic acid conjugates and their free aglycones. The chromatographic system combines the use of a reversed-phase column (LiChrospher CH-18; 5 μm) and a mobile phase of methanol (70–80%)–0.01 M phosphate buffer (pH 6.0) containing 2.5 mM cethexonium bromide as counter-ion at a flow-rate of 1 ml min-1. The hydrophobicity of this quaternary ammonium ion-pairing reagent and the high content of the organic modifier in the mobile phase provide close and short elution times for a wide structural variety of compounds (i.e. alcohols, phenols, steroids, carboxylic acids) and their conjugates with glucuronic acid (capacity factors lower than 7.5), without compromising the selectivity with respect to endogenous compounds of the microsomal incubation medium and urine. Advantages of cethexonium bromide over conventional tetrabutylammonium salts are clearly demonstrated, and the described system was applied to the simultaneous quantitation of clofibric acid and its acylglucuronide in human urine and validated for a pharmacogenetics purpose.