Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback

Water reuse for a number of activities including potable water and replacement of environmental flows is becoming more significant due to the prolonged drought Australia has recently experienced. There is also much debate regarding potential impacts of compounds such as steroid endocrine disrupting chemicals (EDCs), pharmaceuticals and personal care products (PPCPs), and persistent organic pollutants (POPs) to environmental and human health. This paper presents an overview of findings on some EDCs in the Sydney Basin to assess the environmental risk they pose. A tiered approach, using a suite of endpoints spanning in vitro (e.g., estrogen receptor binding assay, the 2-hybrid yeast test) to in vivo (using the mosquitofish (Gambusia holbrooki) to assess vitellogenin induction, and morphological and behavioural changes) studies was conducted on aquatic systems receiving urban and treated sewage effluents. In vitro bioassays suggest low levels of estrogenicity in sewage contaminated waterways. Both estradiol (E2) and estrone (E1) were identified in all river water samples, suggesting that sewage contamination is widespread. The synthetic hormone, ethynylestradiol (EE2), was below detection limits in all samples tested. Results indicate that the STPs were not the only source of EDCs in aquatic systems within the Sydney area. Improvements in treatment technologies in STPs have substantially reduced EDC levels in final effluent as indicated by a reduction inendocrine disrupting effects on the mosquitofish over several years of study. In addition, advanced tertiary treatment technology removed EDCs to levels below that measurable by in vitro assays and in vivo fish testing. This tiered weight of evidence approach provided insights to the risks EDCs in sewage effluent produced from current treatment technologies have on the environment.

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The Endocrine and Reproductive System: Adverse Effects of Hormonally Active Substances?

PEDIATRICS ◽

10.1542/peds.113.s3.1070 ◽

2004 ◽

Vol 113 (Supplement_3) ◽

pp. 1070-1075

Author(s):

Helmut A. Greim

Keyword(s):

Adverse Effects ◽

Intrinsic Property ◽

Reproductive Organs ◽

In Vitro Assays ◽

Endogenous Hormones ◽

Postnatal Exposure ◽

Human Environment ◽

Endocrine Disrupting

Chemicals that have the intrinsic property to modulate or even disrupt the endocrine system are present in the human environment. Because it is the potency of such chemicals that determines the toxicologic relevance, assessment of the risk to human health must consider both the endocrine disrupting potential and the potency. Usually in vitro assays are applied to detect the potential of a hormone-like effect, and such data are considered useful to set priorities for additional testing and for mechanistic studies. However, such data allow only determination of relative potency of a chemical as compared with other xenobiotics, natural compounds, or endogenous hormones. Relevant information on the endocrine-disrupting potency can be taken only from in vivo assays, eg, the Hershberger (male reproductive organs) and uterotrophic (female reproductive organs) assays, the updated versions of the 28- and 90-day toxicity studies in rodents, and the 2-generation studies in rodents. With the use of this information and the concentration of these chemicals in humans, the potency of the effect as compared with endogenous hormone activity can be estimated. So far, the relative potencies of chemicals tested in in vitro systems as compared with estradiol are several orders of magnitude smaller, whereas potency of the phytoestrogen, eg, isoflavones such as genistein or daidzein, can even exceed that of estradiol, especially in infants who are fed soy-based formula as a sole source of nutrition. Although there are still open questions regarding in utero or early postnatal exposure, the low potencies and concentrations of manmade chemicals as compared with the endogenous hormones in humans make it unlikely that adverse effects occur at common exposure.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207321666180220125406 ◽

2018 ◽

Vol 21 (3) ◽

pp. 215-221

Author(s):

Haroon Khan ◽

Muhammad Zafar ◽

Helena Den-Haan ◽

Horacio Perez-Sanchez ◽

Mohammad Amjad Kamal

Keyword(s):

In Silico ◽

Docking Studies ◽

In Vivo Studies ◽

In Vitro Assays ◽

Chronic Obstructive ◽

Lipoxygenase Inhibitors ◽

Lipoxygenase Inhibition ◽

In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

Medicinal Chemistry ◽

10.2174/1573406416666200817160708 ◽

2020 ◽

Vol 16 ◽

Author(s):

Haicheng Liu ◽

Yushi Futamura ◽

Honghai Wu ◽

Aki Ishiyama ◽

Taotao Zhang ◽

...

Keyword(s):

Mammalian Cells ◽

Antimalarial Activity ◽

In Vitro Assays ◽

Compound Library ◽

Antimalarial Agents ◽

Phenotypic Screen ◽

Ic50 Values ◽

Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

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Bioavailability Evaluation of Perchlorate in Different Foods In Vivo: Comparison with In Vitro Assays and Implications for Human Health Risk Assessment

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.1c00539 ◽

2021 ◽

Author(s):

Xin Liu ◽

Hu Zhang ◽

Yimei Tian ◽

Min Fang ◽

Lin Xu ◽

...

Keyword(s):

Risk Assessment ◽

Health Risk ◽

Human Health ◽

Health Risk Assessment ◽

Human Health Risk ◽

Human Health Risk Assessment ◽

In Vitro Assays

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Antibiotic loaded β-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

Scientific Reports ◽

10.1038/s41598-020-80764-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nan Jiang ◽

Devendra H. Dusane ◽

Jacob R. Brooks ◽

Craig P. Delury ◽

Sean S. Aiken ◽

...

Keyword(s):

Tricalcium Phosphate ◽

Calcium Sulfate ◽

Bone Graft Substitute ◽

In Vitro Assays ◽

In Vivo Experiments ◽

Orthopaedic Infections ◽

Clinical Investigations ◽

Antimicrobial Potency

AbstractThis study investigated the efficacy of a biphasic synthetic β-tricalcium phosphate/calcium sulfate (β-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded β-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. β-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded β-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of β-TCP/CS in the management of orthopaedic infections.

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