Extensive Transformation of the Pharmaceutical Carbamazepine Following Uptake into Intact Tomato Plants.

Carbamazepine (CBZ) is an antiepileptic drug which is persistent in wastewater treatment plants and the environment. It has been frequently detected in plant material after irrigation with treated wastewater. To date, little information is, however, available on the transformation of CBZ in plants. In the present study, the uptake, translocation, and transformation of CBZ was studied in hydroponically grown tomato plants. After 35 days of exposure >80% of the total spiked amount of CBZ was taken by the tomato plants and mainly stored in the leaves. A total of 11 transformation products (TP) (mainly phase-I) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and their total amount corresponded to 33% of the CBZ taken up. The ratio of CBZ metabolites to CBZ was highest in fruits (up to 2.5) and leaves (0.5), suggesting an intensive transformation of CBZ in these compartments. Further 10 TPs (phase-I and II) were identified by LC-high resolution mass spectrometry screening, likely comprising another 12% of CBZ. On the basis of these experiments and on an experiment with CBZ-10,11-epoxide a transformation pathway of CBZ in intact tomato plants is proposed that involves epoxidation, hydrolysis, hydroxylation, ring contraction, or loss of the carbamoyl group, followed by conjugation to glucose or cysteine, but also reduction of CBZ. This transformation pathway and analytical data of CBZ transformation products allow for their determination also in field grown vegetable and for the generation of more accurate exposure data of consumers of vegetable irrigated with treated municipal wastewater.

Photochemically Induced Bound Residue Formation of Carbamazepine with Dissolved Organic Matter.

More than 400 new nitrogen containing products were detected upon experimental sunlight photolysis of the pharmaceutical carbamazepine (CBZ) in the presence of dissolved organic matter (DOM) by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). These products were presumably formed through covalent binding of CBZ phototransformation products with DOM molecules. About 50% of these newly formed bound residues contained one nitrogen atom and had a molecular mass between 375 and 525 Da, which was 150 to 200 Da higher than for an average DOM molecule. In addition, a previously unknown CBZ phototransformation product, 3-quinolinecarboxylic acid (3-QCA), was identified by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS). 3-QCA was likely formed through oxidative ring cleavage and subsequent decarboxylation of acridine, a well-known phototransformation product of CBZ. Collision induced dissociation experiments and Kendrick mass defect analyses corroborated that about 160 of the new products were formed via covalent binding of 3-QCA with DOM molecules of above-average O/C and H/C ratios. Experiments at lower CBZ concentration suggested that the importance of bound residue formation increases with increasing DOM/CBZ ratios. Photochemically induced bound residue formation of polar contaminants with DOM in the aqueous phase is thus a disregarded pathway along which contaminants can be transformed in the environment. The method presented here offers a new possibility to study the formation of bound residues, which may be of relevance also for other transformation processes in natural waters where radical intermediates are generated.

Transformation Pathways of the Recalcitrant Pharmaceutical Compound Carbamazepine by the White-Rot Fungus Pleurotus ostreatus: Effects of Growth Conditions.

The widely used anticonvulsant pharmaceutical carbamazepine is recalcitrant in many environmental niches and thus poses a challenge in wastewater treatment. We followed the decomposition of carbamazepine by the white-rot fungus Pleurotus ostreatus in liquid culture compared to solid-state fermentation on lignocellulosic substrate where different enzymatic systems are active. Carbamazepine metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-Q-TOF-MS). In liquid culture, carbamazepine was only transformed to 10,11-epoxy carbamazepine and 10,11-dihydroxy carbamazepine as a dead-end product. During solid-state fermentation, carbamazepine metabolism resulted in the generation of an additional 22 transformation products, some of which are toxic. Under solid-state-fermentation conditions, 10,11-epoxy carbamazepine was further metabolized via acridine and 10,11-dihydroxy carbamazepine pathways. The latter was further metabolized via five subpathways. When (14)C-carbonyl-labeled carbamazepine was used as the substrate, (14)C-CO2 release amounted to 17.4% of the initial radioactivity after 63 days of incubation. The proposed pathways were validated using metabolites (10,11-epoxy carbamazepine, 10,11-dihydroxy carbamazepine, and acridine) as primary substrates and following their fate at different time points. This work highlights the effect of growth conditions on the transformation pathways of xenobiotics. A better understanding of the fate of pollutants during bioremediation treatments is important for establishment of such technologies.

Electrochemistry Combined with LC-HRMS: Elucidating Transformation Products of the Recalcitrant Pharmaceutical Compound Carbamazepine Generated by the White-Rot Fungus Pleurotus ostreatus.

Transformation products (TPs) of environmental pollutants must be identified to understand biodegradation processes and reaction mechanisms and to assess the efficiency of treatment processes. The combination of oxidation by an electrochemical cell (EC) with analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a rapid approach for the determination and identification of TPs generated by natural microbial processes. Electrochemically generated TPs of the recalcitrant pharmaceutical carbamazepine (CBZ) were used for a target screening for TPs formed by the white-rot fungus Pleurotus ostreatus. EC with LC-HRMS facilitates detection and identification of TPs because the product spectrum is not superimposed with biogenic metabolites and elevated substrate concentrations can be used. A group of 10 TPs formed in the microbial process were detected by target screening for molecular ions, and another 4 were detected by screening on the basis of characteristic fragment ions. Three of these TPs have never been reported before. For CBZ, EC with LC-HRMS was found to be more effective than software tools in defining targets for the screening and faster than nontarget screening alone in TP identification. EC with LC-HRMS may be used to feed MS databases with spectra of possible TPs of larger numbers of environmental contaminants for an efficient target screening.

Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.

Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes. Although decision-making can be heavily influenced by environmental cues to induce morphological phenotypes, axial progenitors intrinsically mature over developmental time to favor the neural lineage. Using these data, we present an experimental and analytical framework for exploring the non-homeostatic dynamics of transient progenitor populations as they shape complex tissues during critical developmental windows.

Hijacking of transcriptional condensates by endogenous retroviruses.

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate 'hijacking' of transcriptional condensates in various developmental and disease contexts.

Dnmt1 has de novo activity targeted to transposable elements.

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

Differential regulation of OCT4 targets facilitates reacquisition of pluripotency.

Ectopic transcription factor expression enables reprogramming of somatic cells to pluripotency, albeit with generally low efficiency. Despite steady progress in the field, the exact molecular mechanisms that coordinate this remarkable transition still remain largely elusive. To better characterize the final steps of pluripotency induction, we optimized an experimental system where pluripotent stem cells are differentiated for set intervals before being reintroduced to pluripotency-supporting conditions. Using this approach, we identify a transient period of high-efficiency reprogramming where ectopic transcription factors, but not serum/LIF alone, rapidly revert cells to pluripotency with near 100% efficiency. After this period, cells reprogram with somatic-like kinetics and efficiencies. We identify a set of OCT4 bound cis-regulatory elements that are dynamically regulated during this transient phase and appear central to facilitating reprogramming. Interestingly, these regions remain hypomethylated during in vitro and in vivo differentiation, which may allow them to act as primary targets of ectopically induced factors during somatic cell reprogramming.

Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations.

Balanced chromosomal rearrangements such as inversions and translocations can cause congenital disease or cancer by inappropriately rewiring promoter-enhancer contacts1,2. To study the potentially pathogenic consequences of balanced chromosomal rearrangements, we generated a series of genomic inversions by placing an active limb enhancer cluster from the Epha4 regulatory domain at different positions within a neighbouring gene-dense region and investigated their effects on gene regulation in vivo in mice. Expression studies and high-throughput chromosome conformation capture from embryonic limb buds showed that the enhancer cluster activated several genes downstream that are located within asymmetric regions of contact, the so-called architectural stripes3. The ectopic activation of genes led to a limb phenotype that could be rescued by deleting the CCCTC-binding factor (CTCF) anchor of the stripe. Architectural stripes appear to be driven by enhancer activity, because they do not form in mouse embryonic stem cells. Furthermore, we show that architectural stripes are a frequent feature of developmental three-dimensional genome architecture often associated with active enhancers. Therefore, balanced chromosomal rearrangements can induce ectopic gene expression and the formation of asymmetric chromatin contact patterns that are dependent on CTCF anchors and enhancer activity.

Evaluation of confirmatory data following the Article 12 MRL review for teflubenzuron.

The applicant BASF Agro BV submitted a request to the competent national authority in United Kingdom to evaluate the confirmatory data that were identified for teflubenzuron in the framework of the maximum residue level (MRL) review under Article 12 of Regulation (EC) No 396/2005 as not available. To address the data gaps, a new metabolism study on leafy crops, a study investigating the nature of residues under standard hydrolytic conditions and a validated analytical method to determine residues of teflubenzuron in products of animal origin were submitted. The data gaps were considered satisfactorily addressed. The new information provided does not require a revision of risk assessment performed for teflubenzuron.

Review of the existing maximum residue levels for pencycuron according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance pencycuron. To assess the occurrence of pencycuron residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Commission Regulation (EC) No 33/2008 as well as the European authorisations reported by Member States (including the supporting residues data). Based on the assessment of the available data, MRL proposals were derived and a consumer risk assessment was carried out. Some information required by the regulatory framework was missing and a possible risk to consumers was identified. Hence, the consumer risk assessment is considered indicative only and no MRL proposals were derived by EFSA. Further consideration by risk managers are needed and measures for reduction of the consumer exposure should also be considered.

Setting of import tolerance for quizalofop-P-ethyl in genetically modified maize.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Dow AgroSciences submitted a request to the competent national authority in Finland, to set an import tolerance for quizalofop-P-ethyl in grain from genetically modified maize containing aad-1 gene. The data submitted in support of the request were found to be sufficient to derive a maximum residue level (MRL) proposal for quizalofop-P-ethyl maize grain. Adequate analytical methods for enforcement are available to control the residues of quizalofop-P-ethyl in maize grain. Based on the risk assessment results, EFSA concluded that the authorised use of quizalofop-P-ethyl on genetically modified maize containing aad-1 gene and the subsequent import of maize grain in Europe will not result in a consumer exposure exceeding the toxicological reference value and therefore is unlikely to pose a risk to consumers' health.

Modification of the existing maximum residue level for fluazifop-P in tomato.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Syngenta Crop Protection AG submitted a request to the competent national authority in Portugal to modify the existing maximum residue level (MRL) for the active substance fluazifop-P in tomato. The data submitted in support of the request were found to be sufficient to derive MRL proposal for tomato. An adequate analytical method for enforcement is available to control the residues of fluazifop-P in tomato at the validated limit of quantification (LOQ) of 0.01 mg/kg. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of fluazifop-P according to the reported agricultural practice is unlikely to present a risk to consumer health.

Modification of the existing maximum residue levels for emamectin in leafy brassica and beans and peas with pods.

In accordance with Article 6 of Regulation (EC) No 396/2005, Syngenta France SAS submitted a request to the competent national authority in France to modify the existing maximum residue levels (MRLs) for the active substance emamectin in leafy brassica and beans and peas with pods. The data submitted in support of the request were found to be sufficient to derive MRL proposals for the crops under consideration. An adequate analytical method for enforcement is available to control the residues of emamectin in the commodities under consideration. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of emamectin benzoate according to the reported agricultural practices is unlikely to present a risk to consumer health. The reliable end points, appropriate for use in regulatory risk assessment are presented.

Focussed assessment of certain existing MRLs of concern for acetamiprid and modification of the existing MRLs for table olives, olives for oil production, barley and oats.

In compliance with Article 43 of Regulation (EC) No 396/2005, EFSA received from the European Commission a mandate to provide its reasoned opinion on the existing maximum residue levels (MRLs) for acetamiprid which might lead to consumers intake concerns on the basis of the new toxicological reference values agreed upon by Member States (MSs) in October 2017. In order to identify the MRLs of potential concern that require a more detailed assessment, EFSA performed a preliminary risk assessment, identifying a risk for consumers for 12 commodities. Measures for reduction of the consumer exposure were assessed by EFSA and should be considered by risk managers. Furthermore, in accordance with Article 6 of Regulation (EC) No 396/2005, ADAMA Makhteshim Ltd submitted two requests to modify the existing MRL for acetamiprid in table olives, olives for oil production, barley and oats. The data submitted in support of the requests were found to be sufficient to derive MRL proposals for all crops under assessment. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of acetamiprid according to the intended agricultural practices on table olives, olives for oil production, barley and oats is unlikely to present a risk to consumer health.

Reporting data on pesticide residues in food and feed according to Regulation (EC) No 396/2005 (2017 data collection).

According to Regulation (EC) No 396/2005 on maximum residue levels of pesticides in or on food and feed, Member States have to monitor pesticide residue levels in food samples and submit the monitoring results to EFSA and the European Commission. The Standard Sample Description (SSD, version 1) is the data model used for reporting the data on analytical measurements of chemical substances occurring in food, feed and water to EFSA. This document is a consolidated version of the past 3 years' guidance defining the appropriate SSD codes to describe the samples and the analytical results and it gives directions for the reporting of the pesticide residues monitoring data starting with the data generated in 2017 onwards. These provisions take into account the experience of both the previous reporting seasons and the new legislation applicable in 2017.

Setting of an import tolerance for chlorantraniliprole in hops.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant DuPont de Nemours (Deutschland) GmbH submitted a request to the competent national authority in France to set an import tolerance for the active substance chlorantraniliprole in hops. The data submitted in support of the request were found to be sufficient to derive an maximum residue level (MRL) proposal for hops in support of the authorised use in the USA. Adequate analytical methods for enforcement are available to control the residues of chlorantraniliprole in hops at the validated limit of quantification (LOQ) of 0.01 mg/kg. Based on the risk assessment results, EFSA concluded that the long-term intake of residues resulting from the existing uses and the authorised use of chlorantraniliprole according to the reported agricultural practice is unlikely to present a risk to consumer health. The reliable end points, appropriate for use in regulatory risk assessment are presented.

Modification of the existing maximum residue levels for thiacloprid in corn gromwell seeds and radish.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Nature's Crops International submitted a request to the competent national authority in the United Kingdom to modify the existing maximum residue level (MRL) for the active substance thiacloprid in corn gromwell seeds. Furthermore, the competent national authority in Belgium compiled an application to modify the existing MRL for the active substance thiacloprid in radish. The renewal process for thiacloprid is currently ongoing; in 2015, the Committee for Risk Assessment concluded that the classification as Cat. 1B for adverse effects on development according to CLP criteria is warranted (H360FD). Considering that there is strong evidence that this active substance meets the cut-off criteria for non-approval defined in Article 4 of Regulation (EC) No 1107/2009, further risk management considerations have to be taken into account before a decision on the amendment of the existing MRLs is taken. The data submitted were compliant with the currently applicable legal requirement to derive MRL proposals for corn gromwell seeds and radish. The estimated exposure resulting from the residues of thiacloprid in corn gromwell seeds and radishes is not expected to exceed the toxicological reference values.

Modification of the existing maximum residue levels for clomazone in chamomiles and plantains.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant LSA (Landesanstalt Sachsen-Anhalt) submitted a request to the competent national authority in Germany to modify the existing maximum residue levels (MRLs) for the active substance clomazone in chamomiles and plantains. The data submitted in support of the request were found to be sufficient to derive MRL proposals for the crops under consideration. Adequate analytical methods for enforcement are available to control the residues of clomazone in plant matrices on the crops under consideration at the validated limit of quantification (LOQ) of 0.01 mg/kg. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of clomazone according to the reported agricultural practice is unlikely to present a risk to consumer health. Restrictions on crop rotation as an appropriate risk mitigation measure should be taken into consideration at national level in order to avoid the occurrence of clomazone residues in rotational crops.