Toxicity Assessment of Transfluthrin, Benzyl Butyl Phthalate, and 17ß-Estradiol on the Primary Fibroblast of the Striped Field Mouse, Apodemus agrarius.

Environmental pollution (EP) is a well-known threat to wild animals, but its toxicological impact is poorly understood. In vitro toxicity evaluation using cells of lower predators could be a promising way to assess and monitor the effects of EPs on whole wildlife populations that are related in the food web. Here, we describe EPs' toxic effect and mechanism in the primary fibroblast derived from the embryo of the striped field mouse, Apodemus agrarius. Characterization of the primary fibroblast was via morphology, genetics, immunocytochemistry, and stable culture conditions for optimal toxicity screening. Cell viability assays-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH)-were performed to observe cytotoxicity, and quantitative PCR was conducted to confirm gene alteration by EP exposure. MTT and LDH assays confirmed the cytotoxicity of transfluthrin (TF), benzyl butyl phthalate (BBP), and 17ß-estradiol (E2) with IC50 values of 10.56 µM, 10.82 µM, and 24.08 µM, respectively, following 48-h exposures. mRNA expression of androgen-binding protein, growth hormone receptor, cytochrome C oxidase, and cytochrome P450-1A1 was induced after exposure to TF, BBP, and E2. We unveiled new EP mechanisms at the mammalian cellular level and discovered potential biomarker genes for monitoring of EPs. Based on our findings, we propose the primary fibroblast of A. agrarius as a valuable model to assess the toxicological effects of EP on wildlife.

Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

AIM: Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. METHODS: Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. CONCLUSIONS: The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

Cytotoxicity evaluation of ketoprofen found in pharmaceutical wastewater on HEK 293 cell growth and metabolism.

Inefficient ketoprofen removal from pharmaceutical wastewater may negatively impact the ecosystem and cause detrimental risks to human health. This study was conducted to determine the cytotoxicity effects of ketoprofen on HEK 293 cell growth and metabolism, including cyclooxygenase-1 (COX-1) expression, at environmentally relevant concentrations. The cytotoxic effects were evaluated through the trypan blue test, DNS assay, MTT assay, and the expression ratio of the COX-1 gene. The results of this study show insignificant (p > 0.05) cytotoxic effects of ketoprofen on cell viability and cell metabolism. However, high glucose consumption rates among the treated cells cause an imitation of the Warburg effect, which is likely linked to the development of cancer cells. Apart from that, the upregulation of COX-1 expression among the treated cells indicates remote possibility of inflammation. Although no significant cytotoxic effects of ketoprofen were detected throughout this study, the effects of prolonged exposure of residual ketoprofen need to be evaluated in the future.

In vitro and in vivo assessment of meadowsweet (Filipendula ulmaria) as anti-inflammatory agent.

ETHNOPHARMACOLOGICAL RELEVANCE: Meadowsweet (Filipendula ulmaria (L.) Maxim, Rosaceae) has been traditionally used in most European countries for the treatment of inflammatory diseases due to its antipyretic, analgesic, astringent, and anti-rheumatic properties. However, there is little scientific evidence on F. ulmaria anti-inflammatory effects regarding its impact on cyclooxygenases enzymatic activity and in vivo assessment of anti-inflammatory potential. This study aims to reveal the anti-inflammatory activity of methanolic extracts from the aerial parts (FUA) and roots (FUR) of F. ulmaria, both in in vitro and in vivo conditions. MATERIALS AND METHODS: The characteristic phenolic compounds in F. ulmaria extracts were monitored via high performance thin layer chromatography (HPTLC). The in vitro anti-inflammatory activity of F. ulmaria extracts was evaluated using cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme assays, and an assay for determining COX-2 gene expression. The in vivo anti-inflammatory effect of F. ulmaria extracts was determined in two doses (100 and 200 mg/kg b.w.) with hot plate test and carrageenan-induced paw edema test in rats. Inflammation was also evaluated by histopathological and immunohistochemical analysis. RESULTS: FUA extract showed the presence of rutoside, spiraeoside, and isoquercitrin. Both F. ulmaria extracts at a concentration of 50µg/mL were able to inhibit COX-1 and -2 enzyme activities, whereby FUA extract (62.84% and 46.43% inhibition, respectively) was double as effective as the root extract (32.11% and 20.20%, respectively). Extracts hardly inhibited the level of COX-2 gene expression in THP-1 cells at a concentration of 25µg/mL (10.19% inhibition by FUA and 8.54% by FUR). In the hot plate test, both extracts in two doses (100 and 200mg/kg b.w.), exhibited an increase in latency time when compared with the control group (p<0.05). In the carrageenan-induced acute inflammation test, FUA at doses of 100 and 200mg/kg b.w., and FUR at 200mg/kg, were able to significantly reduce the mean maximal swelling of rat paw until 6h of treatment. Indomethacin, FUA, and FUR extracts significantly decreased inflammation score and this effect was more pronounced after 24h, compared to the control group (p<0.05). CONCLUSIONS: The observed results of in vitro and, for the first time, in vivo anti-inflammatory activity of meadowsweet extracts, provide support of the traditional use of this plant in the treatment of different inflammatory conditions. Further investigation of the anti-inflammatory compounds could reveal the mechanism of anti-inflammatory action of these extracts.

Human fascioliasis endemic areas in Argentina: multigene characterisation of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.

BACKGROUND: In South America, fascioliasis stands out due to the human endemic areas in many countries. In Argentina, human endemic areas have recently been detected. Lymnaeid vectors were studied in two human endemic localities of Catamarca province: Locality A beside Taton and Rio Grande villages; Locality B close to Recreo town. METHODS: Lymnaeids were characterised by the complete sequences of rDNA ITS-2 and ITS-1 and fragments of the mtDNA 16S and cox1. Shell morphometry was studied with the aid of a computer image analysis system. Climate analyses were made by nearest neighbour interpolation from FAO data. Koeppen & Budyko climate classifications were used. De Martonne aridity index and Gorczynski continentality index were obtained. Lymnaeid distribution was assessed in environmental studies. RESULTS: DNA sequences demonstrated the presence of Lymnaea neotropica and L. viator in Locality A and of L. neotropica in Locality B. Two and four new haplotypes were found in L. neotropica and L. viator, respectively. For interspecific differentiation, ITS-1 and 16S showed the highest and lowest resolution, respectively. For intraspecific analyses, cox1 was the best marker and ITS-1 the worst. Shell intraspecific variability overlapped in both species, except maximum length which was greater in L. viator. The desertic-arid conditions surrounding Locality A, the semiaridity-aridity surrounding Locality B, and the very low yearly precipitation in both localities, are very different from the typical fascioliasis transmission foci. Lymnaeids are confined to lateral river side floodings and small man-made irrigation systems. Water availability only depends on the rivers flowing from neighbouring mountains. All disease transmission factors are concentrated in small areas where humans and animals go for water supply, vegetable cultures and livestock farming. CONCLUSIONS: The unusually high number of DNA haplotypes and the extreme climate unsuitable for F. hepatica and lymnaeid development, demonstrate that the transmission foci are isolated. Seasonal transmission may depend on the timely overlap of appropriate temperature and river water availability. Lymnaeids and F. hepatica have probably reached these localities by livestock introduction. DNA differences regarding other populations of L. neotropica and L. viator in Argentina suggest an introduction independent from the spreading movements which allowed these two lymnaeids to expand throughout the country.

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

A new species of Spauligodon (Nematoda: Oxyurida: Pharyngodonidae) in geckos from São Nicolau Island (Cape Verde) and its phylogenetic assessment.

A new nematode species, Spauligodon nicolauensis n. sp., is described from geckos Tarentola bocagei and Tarentola nicolauensis on the island of São Nicolau, Cape Verde. The new nematode was found in the pellets obtained directly from the geckos in a non-invasive fashion, and its identity was assessed both at morphologic and genetic levels. The new species has morphological similarities with Spauligodon tarentolae Spaul, 1926, also parasitizing geckos from the Canary Islands. However, the male cloacal region in the new species is distinct, presenting a different shape of the caudal papillae. The overall resemblance probably resulted from colonization via descent from an ancestor of S. tarentolae carried by the ancestor of Cape Verde Tarentola. The analysis of nuclear DNA sequences confirms that the new species is phylogenetically distinct from all other Spauligodon species already analyzed, forming a group clearly separated from species parasitizing lacertid lizards. The COI genetic distance suggests that the S. nicolauensis n. sp. found in the 2 species of geckos in São Nicolau Island may have resulted from a host-switching event, when they came into contact after the unification of the island.

An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta).

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model 'morphospecies', Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5bp (=0.8%) and interspecific levels were at least 18bp (=c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.

Perspective assessment of COX-1 and COX-2 selectivity of nonsteroidal anti-inflammatory drugs from clinical practice: use of genetic function approximation.

The beneficial action of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with the inhibition of cyclooxygenase-2 (COX-2), whereas their harmful side effects are associated with the inhibition of COX-1. In order to understand a meaningful comparison of both classical NSAIDs and newer COX-2 drugs, a series of molecules from varied classes of COX-2 inhibitors was studied by the application of three-dimensional quantitative structure-activity relationships (3D-QSAR) using molecular descriptors obtained by genetic function approximation. The features responsible for the dual inhibition of COX-1 and COX-2 and the selective inhibition of COX-2 with factors contributing to the maintenance of optimum selectivity were identified. The QSAR models revealed the importance of thermodynamic, electronic, structural, and molecular shape analysis parameters, which can reasonably modulate the selectivity pattern to avoid unsolicited side effects. An improved understanding to rationalize the COX-1 and COX-2 binding profiles could be gained to develop safe drug design methods.

Assessment of celecoxib pharmacodynamics in pancreatic cancer.

Cyclooxygenase-2 (COX-2) inhibitors are being developed as chemopreventive and anticancer agents. This study aimed to determine the biological effect of the COX-2 inhibitor celecoxib in pancreatic cancer as an early step to the further development of the agent in this disease. Eight patients scheduled for resection of an infiltrating adenocarcinoma of the pancreas were randomized to receive celecoxib at a dose of 400 mg twice daily or placebo for 5 to 15 days before the surgery. In addition, carcinomas from nine additional patients were xenografted in nude mice, expanded, and treated with vehicle or celecoxib for 28 days. Celecoxib markedly decreased the intra-tumor levels of prostaglandin E2 in patient carcinomas and in the heterotransplanted xenografts. However, this effect did not result in inhibition of cell proliferation or microvessel density (as assessed by Ki67 and CD31 staining). In addition, a panel of markers, including bcl-2, COX-1, COX-2, and VEGF, did not change with treatment in a significant manner. Furthermore, there was no evidence of antitumor effects in the xenografted carcinomas. In summary, celecoxib efficiently inhibited the synthesis of prostaglandin E2 both in pancreatic cancer surgical specimens and in xenografted carcinomas but did not exert evident antitumor, antiproliferative, or antiangiogenic effect as a single agent. The direct pancreatic cancer xenograft model proved to be a valuable tool for drug evaluation and biological studies and showed similar results to those observed in resected pancreatic cancer specimens.