Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

A large scale mass spectrometry-based histone screening for assessing epigenetic developmental toxicity.

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine).

Active Surveillance of the Safety of Medications Used During Pregnancy.

The scientific community relies on postmarketing approaches to define the risk of using medications in pregnancy because information available at the time of drug approval is limited. Most studies carried out in pregnancy focus on a single outcome or selected outcomes. However, women must balance the benefit of treatment against all possible adverse effects. We aimed to apply and evaluate a tree-based scan statistic data-mining method (TreeScan; Martin Kulldorff, Harvard Medical School, Boston, Massachusetts) as a safety surveillance approach that allows for simultaneous evaluation of a comprehensive range of adverse pregnancy outcomes, while preserving the overall rate of false-positive alerts. We evaluated TreeScan with a cohort design and adjustment via propensity score techniques, using 2 test cases: 1) opioids and neonatal opioid withdrawal syndrome and 2) valproate and congenital malformations, implemented in pregnancy cohorts nested within the Medicaid Analytic eXtract (January 1, 2000-December 31, 2014) and the IBM MarketScan Research Database (IBM, Armonk, New York) (January 1, 2003-September 30, 2015). In both cases, we identified known safety concerns, with only 1 previously unreported alert at the preset statistical alerting threshold. This evaluation shows the promise of TreeScan-based approaches for systematic drug safety monitoring in pregnancy. A targeted screening approach followed by deeper investigation to refine understanding of potential signals will ensure that pregnant women and their physicians have access to the best available evidence to inform treatment decisions.

Teratogenic effects of environmentally relevant concentrations of phenanthrene on the early development of marine medaka (Oryzia melastigma).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in marine environments and have arouse great concern since they pose adverse effects to marine ecosystem. To determine the potential impacts of environmentally relevant PAHs on early life stages of marine fish, this study exposed embryos of marine medaka (Oryzias melastigma) to 0, 2, 10, 50, and 250 µg/L of phenanthrene (Phe), one of the most abundant PAHs. The results demonstrated that Phe exposure decreased hatching rates, delayed hatching time of embryos, and increased deformity rate of newly-hatched larvae. Exposure to 10 and 50 µg/L Phe decreased the survival rate of marine medaka larvae at 28 days post-fertilization (dpf), and no embryo successfully hatched in 250 µg/L Phe exposure group. Morphology results showed that 10, 50, and 250 µg/L Phe exposure significantly retarded the development of embryos, and 2, 10, and 50 µg/L caused yolk sac edema and pericardial edema in newly-hatched larvae, indicating that low concentrations of Phe could induce developmental cardiac toxicity. Furthermore, the changes in the expression of heart development-related genes were determined, and the results showed that Phe-induced cardiac malformation might be related with fgf8, bmp4, smyd1, ATPase and gata4 genes. Overall, environmentally relevant PAHs could disrupt heart morphogenesis and hatching process of marine medaka, which might have profound consequences for sustainability of fish population.

Carcinogenic and teratogenic status of human population and polychlorinated biphenyls contaminations of soils and biota (European pied flycatcher) in a Perm (Western Ural, Russia).

Polychlorinated biphenyls (PCBs) are included in the persistent organic pollutants designated by the Stockholm Convention and are hazardous compounds both for the environment and public health. The aim of this study was to determine the level of environmental pollution of PCBs in the Perm, in soil and biota (European pied flycatcher), and to analyze whether its presence was the cause of cancer and congenital malformations in the population. Soils in the study area had PCB concentrations of 101.87 µg/kg in the Industrialniy District and 27.81 µg/kg in the Leninskiy District of the Perm in 2005. The chemical composition of the individual PCBs and PCB groups were the same in the soils of both regions. The blood of nestlings of the European pied flycatcher raised in the Industrialniy District contained 9.61 ng PCB/ml, while those in the Leninskiy District had 5.64 ng PCB/ml in 2005. A linear correlation was established between the PCB contamination of soils and PCB contamination of pied flycatcher nestling's blood, and inverse linear correlation was established between the content of PCB in the blood of nestlings and the success of breeding of the pied flycatcher in Perm. An epidemiological analysis revealed a high incidence of cancer among the human population of the Industrialniy and Leninskiy districts (371.7 and 376.85 cases per 100,000 population, mean for the 2003-2018, respectively), which exceeded the figure for the whole of Perm (350.77 cases per 100,000 population, mean for the 2003-2018). The incidence rate of congenital malformations in Perm for the study period was 48.51 per 1000 human births. However, a decrease in the concentration of PCBs in soil and biota over a 15-year period (2005-2019) to the less than the detection limit did not lead to a decrease in the incidence of these diseases. Probably, PCB contamination was not the main cause of oncological diseases and congenital malformations in the population of the study area.

Avaliação da toxicidade pré-natal: estudo de teratogenicidade do inseticida piriproxifeno em ratos Wistar / Prenatal toxicity assessment: teratogenicity study of the insecticide pyriproxyfen in Wistar rats

Este estudo investigou a toxicidade pré-natal do inseticida piriproxifeno em ratos Wistar, de forma a detectar possíveis alterações no desenvolvimento fetal da progênie exposta durante o período organogênico. Três doses de piriproxifeno (100, 300 e 500mg.kg-1) foram administradas por via oral às progenitoras, do sexto ao 15º dia de gestação. Os fetos foram submetidos à técnica de diafanização modificada descrita por Taylor e Van Dyke, para avaliação de malformações e alterações esqueléticas. Os resultados não demonstraram a ocorrência de toxicidade materna sistêmica ou alterações nos índices reprodutivos avaliados. Malformações ou alterações teratogênicas não foram detectadas, no entanto alterações esqueléticas sugestivas de retardo no desenvolvimento foram observadas especialmente nas doses mais altas testadas (300mg.kg-1 e 500mg.kg-1). Considerando-se a situação complexa de risco para a saúde humana, mostra-se importante a execução de investigações adicionais, de modo a contribuir para a adequada avaliação de risco do piriproxifeno em água potável.(AU)

This study investigated the prenatal toxicity of the insecticide pyriproxyfen in Wistar rats to detect the possible changes in the fetal development of the progeny exposed during the organogenic period. Three doses of pyriproxyfen (100, 300, and 500mg.kg-1) were administered orally to the progenitors, from day 6 to 15 of gestation. The fetuses were processed using the Taylor and Van Dyke modified diaphanization technique to evaluate malformations and skeletal changes. The results did not demonstrate the occurrence of systemic maternal toxicity or changes in the reproductive indexes evaluated. Malformations or teratogenic changes were not detected, however, skeletal changes suggestive of developmental delay were observed, especially in the highest doses tested (300 mg.kg-1 and 500 mg.kg-1). Owing to the potentially complex situation regarding its risk to human health, it is important that further studies be performed to contribute to the risk assessment of the addition of pyriproxyfen in drinking water.(AU)

Ecological characteristics and teratogenic retinal determination of Cochlodinium geminatum blooms in Pearl River Estuary, South China.

Since 2006, harmful dinoflagellate blooms of Cochlodinium geminatum have infrequently occurred in the Pearl River Estuary, South China. During late October to early November in 2018, C. geminatum blooms occurred again in the region. To investigate the blooming mechanism in certain temporal conditions, we analysed the changes in the environmental parameters and phytoplankton community structure during and after the bloom. The results indicated that the water temperature and salinity had large impacts on the bloom. During the C. geminatum bloom, the phytoplankton community structure changed and the number of dominant species decreased. After the bloom, the species number and abundance of diatoms increased, as the species diversity was recovering. Retinal was detected in the field samples and cultured C. geminatum. It has been demonstrated to exist in some algae species (e.g. Cyanophyta, Chlorophyta, Bacillariophyta, and Euglenophyt), and our results indicates that such teratogens also exist in dinoflagellates. The highest concentration of retinal was detected during the bloom. This result indicates that the retinal content may accumulate during a bloom. Retinal has been demonstrated to be a teratogenic agent and may therefore present a potential risk to aquatic organisms during a bloom episode. This research provided more comprehensive information concerning the ecological influences of C. geminatum blooms.

Bayesian hierarchical methods in the detection of potentially teratogenic first-trimester medications.

PURPOSE: Bayesian hierarchical models (BHMs) have been used to identify adverse drug reactions, allowing information sharing amongst adverse reactions and drugs expected to have similar properties. This study evaluated the use of BHMs in the routine signal detection analyses of potential first-trimester teratogens, where these models have not previously been applied. METHODS: Data on 15 058 malformed foetuses exposed to first trimester medications (1995-2011) from 13 European congenital anomaly (CA) registries were analysed. The proportion of each CA in women taking a specific medication was compared with the proportion of that CA in all other women in the dataset (55 CAs × 523 medications). BHMs were grouped by either medications or CAs or by both simultaneously, and the results compared with analysing each medication-CA combination separately and adjusting for multiplicity using a double false discovery rate (FDR) procedure. The proportions of "high-risk" medications (medications which have been shown to carry a moderate to high risk of foetal malformations) identified as potential signals were compared, as well as the total number of potential signals requiring follow up (the effective workload). RESULTS: BHMs identified more high-risk medications than the double FDR method, but the effective workload was larger. A BHM grouping both medications and CAs, for example, identified 23% of high-risk medications compared with 14% by the double FDR; however, there was an increase from 16 to 71 potential signals requiring follow up. CONCLUSION: For comparable effective workloads, BHMs did not outperform the double FDR, which is comparatively straightforward to implement and is therefore recommended for continued use in teratogenic signal detection analyses.

Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Improving the Safety of Teratogen Prescribing Practices in a Pediatric Rheumatology Clinic.

OBJECTIVES: Although teratogenic medications are commonly used to treat rheumatic disease, no standard model currently exists for educating adolescent patients about teratogenic risk or performing routine pregnancy screening. We performed a quality improvement project to increase education and pregnancy screening in girls and women of childbearing age prescribed teratogenic medications in our pediatric rheumatology clinic. METHODS: Eligible participants included female patients age 10 and older prescribed teratogenic medications in a single-center tertiary care pediatric rheumatology clinic. Seven plan-do-study-act cycles were completed to test the following interventions: visible project reminders, physician and nurse education, progress updates, previsit planning, and development of an electronic health record education template. Chart reviews were performed, and control charts were created for each aim to analyze improvement over time. RESULTS: At baseline, 57 of 231 (24.7%) clinic encounters of female patients age 10 years and older taking teratogenic medications had education documented within the last 12 months, and 47 of 231 (20.3%) had pregnancy screening performed at the visit. Implementation of our interventions resulted in improvement in documentation of annual teratogen education (904 of 1135; 79.6%) and routine pregnancy screening (940 of 1135; 82.8%), both of which were statistically significant (P < .0001). Control charts revealed special cause with sustained improvement over >1 year. CONCLUSIONS: The interventions made through this quality improvement project increased the frequency of both teratogen education and urine pregnancy screening in patients taking teratogenic medications. Development of a standardized education template in the electronic health record played a key role in sustaining these improvements over time.

Approach to evaluating pregnancy safety of anti-rheumatic medications in the OTIS MotherToBaby pregnancy studies: what have we learned?

For the last 30 years, pregnancy exposure studies, with varying methodologies, have been the mainstay of post-marketing surveillance for new drugs likely to be used by women of reproductive age. While they provide valuable data to inform use during pregnancy, they have limitations that render them necessary but not sufficient in supplying timely information to patients and prescribers. The Organization of Teratology Information Specialists MotherToBaby Pregnancy Studies' collaborative research group operates to help fill this gap. This paper provides an overview of the research that has been and is currently being conducted, as well as best practices determined over the past two decades. The Organization of Teratology Information Specialists MotherToBaby studies can provide earlier signaling with regard to concerns following possible teratogenic exposures, which when examined in conjunction with larger database studies and case-control designs, can move us closer to developing a fuller picture of drug safety for women of reproductive age.

Animal Embryotoxicity Studies of Key Non-Artemisinin Antimalarials and Use in Women in the First Trimester.

The World Health Organization currently recommends quinine+clindamycin for use against malaria in the first trimester. This may soon change to recommending artemisinin-based combination therapies (standard duration of dosing = 3 days). The non-artemisinin partner drugs include amodiaquine, lumefantrine, mefloquine, piperaquine, sulfadoxine+pyrimethamine, and pyronaridine. For quinine, clindamycin, and mefloquine and the combinations of sulfadoxine+pyrimethamine and artemether+lumefantrine, there are reports (including studies without internal comparison groups) that combined describe 304 to >1100 exposures of women in the first trimester for each drug with no conclusive evidence of adverse effects on pregnancy at therapeutic doses. This is despite the fact that all of these drugs or drug combinations caused embryo deaths and/or malformations in at least one animal species and all except lumefantrine had at least one exposure ratio <1. It now seems that these animal studies overestimated the risk of developmental toxicity in women with malaria. Three other non-artemisinins (amodiaquine, piperaquine, and pyronaridine) have few or no reported exposures in women in the first trimester and have exposure ratios ≤2 based on studies in pregnant rats and rabbits with dosing throughout organogenesis. However, none of these drugs caused embryo deaths or malformations in pregnant rats and rabbits with the exception of pyronaridine, which caused embryo deaths only at a dose that was excessively toxic to the mothers. Thus, for amodiaquine, piperaquine, and pyronaridine, the testing in animals did not reveal findings of concern and the exposure ratios were in the range of the other non-artemisinin antimalarials described above. Birth Defects Research 109:1075-1126, 2017. © 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.

Using passive sampling and zebrafish to identify developmental toxicants in complex mixtures.

Using effects-directed analysis, we investigated associations previously observed between polycyclic aromatic hydrocarbons (PAHs) and embryotoxicity in field-deployed low-density polyethylene (LDPE). We conducted effects-directed analysis using a zebrafish embryo assay and iterative fractionation of extracts of LDPE that were deployed in the Portland Harbor superfund megasite, Oregon (USA). Whole extracts induced toxicity including mortality, edema, and notochord distortion at 20% effect concentration (EC20) values of approximately 100, 100, and 10 mg LDPE/mL, respectively. Through fractionation, we determined that PAHs at concentrations similar to previous research did not contribute markedly to toxicity. We also eliminated pesticides, phthalates, musks, and other substances identified in toxic fractions by testing surrogate mixtures. We identified free fatty acids as lethal components of LDPE extracts and confirmed their toxicity with authentic standards. We found chromatographic evidence that dithiocarbamates are responsible for notochord and other sublethal effects, although exact matches were not obtained. Fatty acids and dithiocarbamates were previously unrecorded components of LDPE extracts and likely contribute to the toxicity of the whole mixture. The present study demonstrates the success of effects-directed analysis in nontargeted hazard identification using the zebrafish embryo test as a self-contained battery of bioassays that allows identification of multiple chemicals with different modes of action. This is the first effects-directed analysis to combine LDPE and zebrafish, approaches that are widely applicable to identifying developmental hazards in the bioavailable fraction of hydrophobic organic compounds. Environ Toxicol Chem 2017;36:2290-2298. © 2017 SETAC.

Developmental toxicity in flounder embryos exposed to crude oils derived from different geographical regions.

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees.

Developmental and reproductive toxicity of PVP/PEI-coated silver nanoparticles to zebrafish.

Cellular and molecular mechanisms of toxicity of silver nanoparticles (NPs) and their toxicity to fish embryos after waterborne exposure have been widely investigated, but much less information is available regarding the effect of Ag NPs on physiological functions such as growth or reproduction. In this work, the effects of waterborne exposure of adult zebrafish (Danio rerio) to PVP/PEI coated Ag NPs (~5nm) on reproduction (fecundity) were investigated. Moreover, the development of the embryos after parental exposure was compared with the development of embryos after direct waterborne exposure to the NPs. For this, two experiments were run: 1) embryos from unexposed parents were treated for 5days with Ag NPs (10µgAgL-1-10mgAgL-1) and development was monitored, and 2) selected breeding zebrafish were exposed for 3weeks to 100ngAgL-1 (environmentally relevant concentration) or to 10µgAgL-1 of Ag NPs, fecundity was scored and development of resulting embryos was monitored up to 5days. Waterborne exposure of embryos to Ag NPs resulted in being highly toxic (LC50 at 120h=50µgAgL-1), causing 100% mortality during the first 24h of exposure at 0.1mgAgL-1. Exposure of adults, even at the environmentally relevant silver concentration, caused a significant reduction of fecundity by the second week of treatment and resulting embryos showed a higher prevalence of malformations than control embryos. Exposed adult females presented higher prevalence of vacuolization in the liver. These results show that Ag NPs at an environmentally relevant concentration are able to affect population level parameters in zebrafish.

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study.

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

The Hydra regeneration assay reveals ecological risks in running waters: a new proposal to detect environmental teratogenic threats.

The regenerative ability of Hydra vulgaris was tested as potential biomarker for the development of a new eco-toxicological index. The test is based on the regeneration rate and the aberration frequency of the columna (body and adhesive foot) after separation from head and tentacles by a bistoury. Particularly, 45 columnae were submerged in the rearing solution (that is Hydra medium) to have control, and 285 in potential contaminated waters to have treatments, collected from 19 sites along 10 rivers in central Italy. ANCOVA and chi-square tests were used to compare values from each site to a laboratory control. Subsequently the values on regeneration rate and aberration frequency were inserted in a double entry matrix, where the match of the two entries in the matrix provides the score of the proposed Teratogenic Risk Index (TRI). Each score corresponded to one of the 5 teratogenic risk classes, to which a risk level was associated: from 1 (no risk) to 5 (very high risk). On the whole, 32% of the studied sites were classified as no teratogenic risk while the remaining showed a variable risk level from low to very high. This study proposed for the first time an early warning system to detect the presence of teratogens in running waters, providing a rapid and cost-effective evaluation method. Therefore, TRI may contribute to initiate adequate measures to manage riverine habitats, and to monitor the running water teratogenic status. Specifically, this index may provide the opportunity to identify the disturbance sources and then to drive the decisions, together with competent authorities, on the catchment and landscape management and on the possible use of waters for urban, agricultural, and industrial activities, since they may show significant effects on the human health.

Characterization of teratogenic potential and gene expression in canine and feline amniotic membrane-derived stem cells.

The biosafety of innovative procedures that utilize stem cells in regenerative medicine has been addressed in several studies. Previous work has showed no tumour formation following the use of feline and human amniotic membrane-derived stem cells (AMSCs). In contrast, tumour formation was observed when canine AMSCs were utilized. These findings suggested that feline and human, but not canine, AMSCs are suitable for cell transplantation trials. This study aimed to further evaluate the feasibility of utilizing canine AMSCs for transplantation purposes as well as for felines. We tested teratoma formation following cell injection into BALB/c nude mice and then assessed expression of haematopoietic, mesenchymal, tumorigenic, pluripotency and cellular regulation markers using flow cytometry and qPCR. The use of canine AMSCs did not result in macroscopic tumour formation as determined 60 days after transplantation. The immunophenotypic characterization by flow cytometry revealed expression of mesenchymal markers (CD73 and CD90) and expression of the pluripotent marker OCT4 and SOX2. Quantitative PCR analysis revealed that there were no differences in the patterns of gene expression (CD34, CD73, OCT4, CD30 and P53) between canine and feline AMSCs, with the exception of the expression of SOX2 and CD90.

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition.

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer's storage conditions. Sample extraction was performed using a liquid-liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g(-1). AME levels ranged between 11 and 137 ng g(-)(1), which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.