Median particle number concentrations were 4.8 × 104, 1.3 × 105, and 5.4 × 105 particles/cm3, respectively. Nanoparticles accounted for 95, 40, and 61%, respectively, with prevailing Fe and Mn. All markers of nucleic acid and protein oxidation, malondialdehyde, and aldehydes C6–C13 were elevated, already in the pre-shift samples relative to controls in both workshops. Significant post-shift elevations were documented in lipid oxidation markers. Significant associations were found between working in nanocomposite synthesis and EBC biomarkers. More research is needed to understand the contribution of nanoparticles from nanocomposite processing in inducing oxidative stress, relative to other co-exposures generated during welding, smelting, and secondary oxidation processes, in these workshops.Researchers in nanocomposite processing may inhale a variety of chemical agents, including nanoparticles. This study investigated airway oxidative stress status in the exhaled breath condensate (EBC). Nineteen employees (42.4 ± 11.4 y/o), working in nanocomposites research for 18.0 ± 10.3 years were examined pre-shift and post-shift on a random workday, together with nineteen controls (45.5 ± 11.7 y/o). Panels of oxidative stress biomarkers derived from lipids, nucleic acids, and proteins were analyzed in the EBC. Aerosol exposures were monitored during three major nanoparticle generation operations: smelting and welding (workshop 1) and nanocomposite machining (workshop 2) using a suite of real-time and integrated instruments. Mass concentrations during these operations were 0.120, 1.840, and 0.804 mg/m The global market for nanocomposites is expected to reach over $5 billion by 2020, and it is fueled by demand in numerous sectors, from automotive to military and aerospace applications . ResearcVolatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs), generated as a result of polymer heating and thermal degradation, polycyclic aromatic hydrocarbons (PAHs), and other byproducts of chemical reactions with ozone and catalytic metal oxides used as fillers, may lead to complex airborne pollutants. Lower molecular weight PAH vapor may be converted to higher molecular weight PAHs and other compounds via catalytic reactions with metal oxide fillers . CondensSince inhalation remains the major exposure pathway to nanoparticles during nanocomposite manufacturing, the respiratory system is the main portal of entry and the primary target organ of concern. However, there are only a few sensitive non-invasive methods to probe deep airways in humans. Exhaled breath condensate (EBC) collection and analysis is one such technique. EBC is a liquid that reflects the composition of the fluid lining the airway . It is o2+ flux, induction of pro-inflammatory mediators through receptor stimulation, hypersensitivity, genotoxicity, and cell necrosis [A large body of in vivo and in vitro particle toxicology and nanotoxicology studies have shown that nanoparticles induce toxicity via a number of mechanisms, including intracellular reactive oxygen species (ROS) generation and ensuing oxidative stress, Canecrosis ,16,17,18necrosis ,20,21. Hnecrosis . iso-Prostaglandin F2α (8-isoprostane) is produced by free-radical lipid peroxidation of arachidonic acid, and represents an in vivo specific marker of oxidative stress. Oxidative modification of lipids occurs in vivo during aging and in certain disease conditions. Lipid peroxides are unstable indicators of oxidative stress in cells that form more complex and reactive compounds, such as malondialdehyde (MDA), 4-hydroxy-trans-hexenal (HHE), and 4-hydroxy-trans-nonenal (HNE), that can form covalent adducts with biomolecules including DNA and proteins, and thus, are regarded as genotoxic and cytotoxic [−) and hypochlorous acid (HClO), respectively, with tyrosine residues of proteins, which may lead to functional relationship with the neutrophilic inflammation. Both 3-NOTyr and o-tyrosine (o-Tyr) have been found in patients with interstitial lung diseases, but studies on other biomarkers are limited. Oxidative damage to nucleic acids may be measured using 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) formed by oxidation of guanine from DNA and 5-hydroxymethyl uracil (5-OHMeU) from RNA [A common feature of many aerosols in the workplaces is their ability to generate ROS, which induce oxidative damage to biomolecules, leading to activation of redox signaling pathways. 8-ytotoxic . 3-Nitrofrom RNA ,23.The first aim of this study was to characterize workplace aerosols generated during synthesis and post-processing of nanocomposites, and associated operations, including machining of nanocomposite materials, smelting, and welding. Another aim was to measure markers of oxidative stress in the EBC of workers exposed to nanoparticles and to study their possible association with workplace environments. This is the first study to report on the respiratory health status on this cohort of researchers and applicators, using a suite of well-established biomolecular markers measured in the EBC. In a separate paper, we report on inflammatory leukotrienes (B4 and cysteinyl leukotrienes), pro-inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF), interleukins , and their anti-inflammatory counterparts , in the EBC of workers, as well as fractional exhaled NO, and lung function using spirometry . The research and development unit at a national research university is trying to develop a new thermoplastic or reactoplastic (thermoset) composite material that exhibits comparable performance characteristics with steel, with regard to its low thermal expansion, hardness, and resistance to surface scratching. This new nanocomposite material is intended for use in automated measuring devices of plastic materials, where metal instruments cannot be used because of measurement errors related to their higher expansion and the risk of scratching the surface of plastics. Researchers would normally perform three different operations: welding on metal surfaces, smelting of mixtures containing nanoadditives, and machining of the finished nanocomposite. The researchers were divided into two separate groups in two workshops. Welding and smelting took place together in workshop 1, located in the basement of the building, whereas composite machining took place in a second workshop located on the ground floor of the same building. Researchers would usually carry on their daily operations in the workshops for about 2 h, with the majority of the working day spent in their offices. The three operations would occur simultaneously in both workshops, and be performed by different individuals.2 in epoxide resins.On the day of examination, the duration of each of these (simultaneous) operations lasted about 150 min, and the original source of a nanocomponent was amorphous colloidal nanoSiO2 mixed-copper coated wire (G3Si) and coated electrodes EB 121 . The S355J2 steel content was (in wt %): Fe, 97.39; C, 0.24; Mn, 1.70; Si, 0.6; P, 0.035; S, 0.035). Also in workshop 1, an AlSi9Cu3 alloy , mixed with modifying salts , was smelted in the smelting oven at 760 °C, in a mold made of sand mixed with a bentonite filler.Welding on metal surfaces (workshop 1) was conducted on mild steel S355J2 using metal active gas (MAG) technology with Ar/COMachining of surfaces of previously finished nanocomposite blocks, including milling, grinding, and polishing, was performed in workshop 2. Mixing of the fillers with matrices to prepare nanocomposites samples was performed about twice per month, and lasted about 2 h.2 fillers. One nanocomposite specimen contained 1.0% w/w amorphous colloidal nanoSiO2, whereas the second specimen contained crushed rice husks with 50% cellulose, 30% lignin, and up to 20.0% SiO2 as fillers. Three additional samples were geopolymers, first containing metakaolin mixed with NaOH only, and two others were filled with ash or basalt, each at 40% w/w. The formulations of nanocomposites differed over time, according to research aims. On other days, the operations may include thermoplast melting (polypropylene) and mixing with milled coconut fibers, composite materials production, or adding textile fibers, glass, etc.On the day of examination, five nanocomposite specimens were processed on workshop 2. Two samples contained epoxide resins with SiOEBC samples were collected from the 19 nanocomposite-synthesizing and processing researchers  in September 2016. On the day of examination, eleven researchers were working in workshop 1 (welding and smelting), and seven in workshop 2 (machining of nanocomposites). The control group was composed of 19 subjects  from the same town, not employed in this plant nor occupationally exposed to dust or other health risks. Exposed and control subjects were administered a standardized questionnaire that collected information on personal and occupational history, medical treatments, dietary habits, smoking habits, and alcohol intake. Participants underwent a physical examination in another part of the building, followed by the collection of their EBC. Exclusion criteria for all subjects were history of tuberculosis, myocarditis, congenital heart disease, lung cancer, and recent fever and/or common cold symptoms. Nanocomposite researchers provided EBC both pre-shift  and post-shift . For simplicity, we refer to these examinations as pre-shift and post-shift, even though the remainder of their total 8-h shift was spent in their offices. Among the workers, seven were working in the same workshop as usual, and the remaining twelve were working in both workshops during the previous weeks. The controls provided EBC only once at approximately the same time of day as researchers in the workshop floor. The pre-shift samples were used to study the subacute/chronic effect on the subjects resulting from exposures in previous days. Comparison of the pre-shift and post-shift samples was intended to evaluate the acute effect of exposure during the shift.The study was conducted according to the Declaration of Helsinki. The Ethical Committee of the 1st Medical Faculty, Charles University, approved the study. All participants were informed of the study aim at least five days earlier, and signed an informed consent form before the study began.Chemical and physical characterization of nanoparticles and aerosols was carried out using a set of real-time and size selective aerosol instruments. A Berner low-pressure impactor  ,26 was uThe online real-time instrumentation included two standard aerosol spectrometers—a scanning mobility particle sizer SMPS 3936L  and an aerodynamic particle sizer APS 3321 , covering the size range of aerosol particles from 6 nm up to 20 µm. In addition, an ultrafine condensation particle counter UCPC 3025  was used to measure the total particle number concentration (10 nm–~1 µm) and three optical particle sizers OPS 3330  measuring number size distribution in the range 300 nm–10 µm. All instruments sampled for the whole duration of the current nanoparticle generation operations . Background nanoparticle concentration was monitored before the operations started. The online instruments were placed about 1.5 m from the workers at a height of approximately 1.5 m. The details concerning the samples collected are shown in A scanning electron microscope (SEM) Indusem, , equipped with energy-dispersive X-ray (EDX) spectroscopy , was used for elemental analyses and EDX spectra acquisition at an accelerating voltage of 15 kV for 120 s. Carbon sputtering was used for deposition of a conducting layer (ca. 15 nm thick) onto the samples, to prevent charging. To cover a representative area and minimize the effects of possible heterogeneities, seven square areas containing aerosol with an edge length of 200 μm were scanned on each sample. Carbon, oxygen, and copper were excluded from the results as they were present in the substrates, superficial conducting layer, and The data from EDX were recalculated to mass concentration using sulfur as internal reference, and sulfate mass concentration from IC recalculated to sulfur under the assumption that all sulfur in the sample was present as sulfates. Control recalculations were done using chlorine as reference and chloride mass concentration from IC, with very similar results as for sulfur.EBC samples were collected using an Ecoscreen Turbo DECCS device , that was equipped with a filter. All subjects breathed tidally through a mouthpiece connected to the condenser (−20 °C) while wearing a nose-clip. A minimum volume of exhaled air of 120 L was maintained , and the time of collection was about 15 min . All sam6–C13, and 8-isoprostane; nucleic acids—8-OHG, 8-OHdG, and 5-OHMeU; and protein-o-Tyr, 3-ClTyr, and 3-NOTyr. The α-amylase concentration was monitored [A panel of oxidative stress markers derived from free radical oxidation of polyunsaturated fatty acids, nucleic acid bases, and proteins was analyzed after solid-phase extraction (SPE) by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) using deuterium-labelled internal standards, as previously described ,29,30. Tonitored . Electrionitored . All sam2, O3, nitrogen oxides (NOx), particulate matter (PM)2.5 and PM10, recorded on an hourly basis, were taken from the monitoring data on the same days when the workers were examined. Air concentrations were obtained from the National Hydrometeorological monitoring system at the closest stationary monitoring station. The distance to the site of EBC collection was about 3.0 km. Environmental monitoring employed the following analytical methods: UV-fluorescence (SO2), chemiluminescence (NOx), UV-absorption (O3), and an optoelectronic method (PM). Air concentrations of SOt-test  and the Mann-Whitney U test . The paired sample t test  was used to compare workers’ pre-shift and post-shift values of oxidative stress markers. The bivariate relationship between variables under study was assessed using the Spearman correlation coefficient. Multiple regression analysis was used to predict markers in EBC by a set of predictors . Statistical significance was set at p < 0.05. All analyses were conducted using SPSS version 22.0 .Basic descriptive statistics  were computed for all variables, which were subsequently tested for normality using the Kolmogorov–Smirnov test. The chi-square test was used to compare frequency counts of demographic categorical variables  in groups of workers vs. controls. Differences in interval variables were tested using independent-group p > 0.05).Demographic characteristics of the exposed and control groups are presented in Personal protective equipment was used for welding  and smelting (gloves). No respiratory protection was used during any of the operations.5 particles/cm3 (the interquartile range (IQR) was from 1.2 × 105 to 1.5 × 105 particles/cm3) and maximum number concentration reached about 2.1 × 105 particles/cm3. However, these particles were formed by coagulation of primary particles smaller than 10 nm with high concentrations, as was observed from the time evolution of number concentrations and size distributions. While concentrations of the lowest size bins were quickly decreasing, concentrations of accumulation mode particles steadily increased.MAG welding resulted in monomodal particle size distribution, with particles mainly in the accumulation size range, with modal size around 200 nm. Total median particle number concentration was 1.3 × 104 particles/cm3 (IQR 3.1 × 104 to 9.0 × 104 particles/cm3), and the highest concentration reached about 2.3 × 105 particles/cm3. The time evolution of larger size bins showed a significant contribution of the smallest size bin to the total number concentration for almost the whole sampling period, except the beginning of the measurement, before the smelting vessel was put into the oven.Particle size distributions produced by smelting were multimodal. The first mode, <25 nm, was formed by the primary nanoparticles originating from evaporation of the material being smelted and its subsequent condensation. The second mode, around 50 nm, was a product of coagulation of the primary particles. The third mode, with maxima around 200 nm, was most probably coming from the previous operation, MAG welding, where the particles in the workplace did not have enough time to deposit. Most of the particles produced by smelting were released when the smelting oven was open. The measurement after the smelting was finished showed a change of the size distribution shape into unimodal, and the shift of the mode of primary particles to about 25 nm as a product of coagulation. Total median particle number concentration was 4.8 × 105 particles/cm3 (IQR 3.1 × 105 to 6.8 × 105 particles/cm3), with maximum concentration around 8.8 × 105 particles/cm3.Operations of mechanical grinding and milling of various nanocomposites produced primary larger particles >1 µm, but also, nanoparticles produced by condensation in the colder air far from the machining tool of semivolatile organic compounds and thermal breakdown products generated form localized heating of the polymer during machining. In this case, the measured size distribution exhibited unimodal shape, with a maximum at about 100 nm with a large tail for supermicron particles. It seems that these particles may have represented the primary particles produced by grinding and milling. Total median particle number concentration was 5.4 × 10Nevertheless, comparison of the process data with the background measurement in the same workshop floor suggests contribution of other sources of particles. Time plots of the larger size bins revealed that the highest contribution to the total number concentration was from the accumulation mode particles (100 nm–1 µm).All the measurements of the background values showed high particle concentrations, which might have considerably influenced the measurements, making the estimation of the contribution of individual sources very complex. The background values were measured first during the night in the corridor next to the workshops. Then, before each working operation, 15 min of measurements were taken as background values. There were no other obvious sources of aerosol background particles, except from indoor penetration of outdoor particles. Particle number size distributions during the operations are shown in The percentage of total particulate number concentrations (<10 µm) in larger size bins, measured by SMPS and APS (based on aerodynamic diameter) is shown in 3, the second highest during machining, with 0.804 mg/m3, and the lowest during smelting, with 0.120 mg/m3. Fine mode peaked at around 300 nm in aerodynamic diameter during welding and smelting, when the mass size distributions were almost monomodal, while during machining it peaked at about 200 nm with bimodal mass size distribution, as can be seen in The highest mass concentration measured by BLPI was found during welding, with mean 1.840 mg/mIn the case of welding, the particles were dominated by iron, manganese, and silicon, with manganese and silicon being enriched in comparison with welded material. The maximum of size distribution was between 247 and 435 nm in aerodynamic diameter both for manganese and iron. Silicon concentration was the largest for the biggest analyzed particles.Smelting operation was done in the same workshop as welding, and despite intensive venting after the previous operation, the analyzed aerosol captured most of signatures of particles from welding. Therefore, the particles were again dominated by iron, manganese, and silicon. Some influence of smelting was visible, especially on last stage, with size range 25–56 nm where aluminum, sodium, and chloride were enriched. The mass size distribution had the same features as from welding. Due to an approximately 30× dilution of aerosol from welding, some influence of ambient aerosol was visible, especially based on sulfur concentrations.Elemental composition of size-resolved aerosol fractions during the three operations are shown in The data from machining of geopolymers exhibited the largest variability in elemental composition, suggesting a more complex range of sources. Moreover, the total elemental analyzed mass was only a few percent of the total mass, while in previous operations, it accounted for ~50% of the total mass.2O3, SiO2, TiO2, Fe2O3, MnO2). Based on our experience from similar past experiments, the missing mass was most probably composed of carbonaceous matter. In the nanofraction, i.e., lowest two impactor stages with particle aerodynamic diameters 25–56 and 56–100 nm, the missing mass formed over 95% of the total mass.We have attempted to calculate the missing mass as a difference between the total mass determined gravimetrically, and sum of all analyzed ions + Al, Si, Ti, Fe, Mn, where analyzed elements were represented by their oxides . No significant decrease was found in EBC markers with increasing latency from last exposure. Similarly, no difference was found between researchers who switched workshops in the previous days or weeks  between production and machining of nanocomposites in nine pre-shift and nine post-shift markers, as shown in Multiple regression analysis confirmed a significant association , the main proportion of nanoparticles in workplace aerosol originated during machining and smelting of the samples using abrasive structures to modify the surface of the products or at hot temperature. Iron represented the highest proportion of elements detected in the aerosol during all operations monitored, including machining. Similarly, when the elements were ranked according to their mass concentrations in the nanosized fractions (last two impactor stages), Fe was the more abundant element, followed by Mn, Si, Al, S, Na, Cl, and K. This means that even though nanoSiO2 was added in substantial amounts into the machined polymeric materials, its release into the form of nanosized aerosol particles was not as significant. We presume that most of the aerosol mass during machining operation was likely composed of organic compounds, as shown in several of our previous studies [Although production of the nanocomposites in this research plant included the addition of nanoparticles  and exposures in the workshop were longer  [3 and 6.7 × 104 particles/cm3, respectively. The results of most markers of oxidation of lipids, nucleic acids, and proteins in the Fe oxide study were 110–120% higher than in nanocomposite researchers [The results of elevated markers of oxidative stress agree with our previous findings in other production plants, where workers were exposed to poorly soluble engineered metal oxide nanoparticles. The highest levels of lipids, nucleic acids, and protein markers of oxidative stress reached about 130% of the values reported here . In thosanoTiO2) . The lenearchers . 2 and iron oxide manufacturing workers, but the duration of the operations associated with nanoparticle exposures was shorter, typically <2 h/day, with the remainder of the shift spent in offices in another part of the building. Another important consideration is that in previous factories, the proportion of the nano-sized particles in the inhaled aerosol was high (80–85%), whereas in the current study, it was lower and more variable (40–95%). Even in the office, in the employees who visited the TiO2 production area and were exposed to the aerosol for an average of 14 min/day, the EBC markers reached 50–60% of the levels measured in TiO2 production workers, which was significantly higher when compared to controls [The aerosol mass and number concentrations were higher in this study of nanocomposite workers than in our earlier studies of nanoTiOcontrols ,42. No association was seen with lifestyle factors or environmental air contamination in any of the studies, including this one.2 particles were identified in the pre-shift EBC samples of the workers [3 nanoTiO2 in rats over 2 weeks , histopathological lesions persisted at post-exposure day 7, but resolved at day 15 [Of note, EBC markers of oxidative stress in this study were already elevated in pre-shift samples, suggesting chronic effects. Accordingly, in our previous study, the pre-shift markers were elevated , and TiO workers . The biot day 15 . 2, SiO2, or indium tin oxide). In a recent study of workers exposed to high levels of nanoTiO2, Zhao et al. (2018) documented elevated cardiopulmonary disease markers, impairment of lung function, X-ray interstitial changes, and elevated blood MDA and TNF biomarkers that were associated with occupational exposure [Furthermore, other human studies brought results supporting the potential of nanoparticles to produce oxidative stress in human volunteers ,47,48,49exposure . In the exposure . This isexposure . One major limitation of this study is the small number of subjects, which unfortunately, is limited by the small size of this workforce, as all available research workers were included. This is a common issue in epidemiological studies of nanomanufacturing workers because the industry is still evolving, consolidating, and changing all the time. Secondly, aerosol measurements were based on area sampling, not personal exposures, as good quality personal nanoparticle samplers were not available at the time of this field study. Another challenge is the mixed nature of exposures, which makes it difficult to discern effects resulting from engineered nanoparticles, incidental ones, nanocomposite synthesis and processing, or auxiliary operations such as welding. As has already been mentioned, other substances specifically generated in combustion processes, such as welding and smelting, like e.g., ozone, carbon monoxide, and polycyclic aromatic hydrocarbon, are likely to be causal for the effects analyzed. Due to limited access to the workplace, we could only collect a relatively small number of measurements. We acknowledge limited characterization of exposure variability in the current study. Although aerosol exposures were well characterized for each operation, day-to-day and seasonal variability due to variations in operations, working conditions, and activities, requires repeated measures. Furthermore, we do not have a good explanation for the high background exposure levels measured in some cases, although persisting nanoparticles in background air from past working operations cannot be excluded. This study is ongoing, and these limitations will be addressed in follow-up visits. Exposure sources in individual workshops/laboratories were usually mixed, and it is difficult to apportion aerosol source contribution to the overall nanoparticle exposures. o-Tyr and 3-NOTyr from proteins; and MDA, and ALD6-12 from lipids. We will also keep 8-isoprostane, a biomarker that differentiated the subgroups of researchers (higher in workshop 2 during machining of nanocomposites) in subsequent investigations. A notable strength of our study is the large spectrum of biomarkers and clinical endpoints that were examined. Consistency in study design and biomarker endpoints across several studies of nanoparticle-exposed workers further enabled us to examine relationships between pre-shift and post-shift biomarkers in EBC, and identify the more robust biomarkers for biomonitoring purposes. Due to frequent correlation between the pre-shift and post-shift markers in this study, we could limit EBC collection to the post-shift samples. Furthermore, by exploring the patterns of biomarker from our earlier studies and the strength of association in multiple regression analysis, we could narrow the broad spectrum of markers of oxidation to the following markers that appeared to be the most robust set of markers across all our studies: 8-OHdG and 5-OHMeU from nucleic acids; One major challenge with sensitive biomonitoring techniques is their limited specificity towards exposure triggers and the difficulty in interpreting the meaning of their physiological values in the context of chronic disease development and damage-repair kinetics. It is likely the transition metals, especially Fe and Mn and their dissolved ions, contribute significantly to oxidative stress in lung tissues ,34. SimiOur results are consistent with the oxidative stress hypothesis, and suggest lung injury at the molecular level. Researchers in nanocomposite processing had elevated levels of oxidative stress markers in EBC. In a companion manuscript, we also report on elevated levels of inflammatory markers in EBC and chronic bronchitis among this cohort of workers. These effects are likely caused by mixed exposures to aerosols, originating from welding, smelting, and nanocomposite processing. More research is needed, as other substances generated in combustion processes, including welding and smelting, like e.g., ozone, or polycyclic aromatic hydrocarbons in both workshops, should be evaluated. 2 manufacturing workers who were exposed for longer operation durations over similar periods of time. Considering that exposures in this study were long-term (close to two decades), but of short duration and frequency , chronicity of such short exposure episodes is sufficient to trigger chronic oxidative stress and inflammatory responses comparable to the levels of EBC biomarkers found in (nano)Fe oxide nanomanufacturing workers, and of the same order of magnitude  than in nanoTiOWe can strongly recommend using both the ventilation system in the workshops and the personal protection equipment of the researchers. o-Tyr, 3-NOTyr, MDA, ALD6-12, and 8-isoprostane as the most sensitive and robust biomarkers across all our EBC studies.Analysis of exhaled breath has immense potential to bring data enabling monitoring of several exposures from the workplace and the environment where nanoparticles and other inhalable toxicants may play a significant role. We recommend post-shift analysis of the following markers: 8-OHdG, 5-OHMeU,


Coryphantha macromeris (Engelm.) Britton & Rose growing under greenhouse conditions was qualitatively investigated for the first time by means of modern ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). The UHPLC-PDA-HESI-Orbitrap-MS/MS analysis indicated a high complexity in phenolic metabolites. In our investigation, 69 compounds were detected and 60 of them were identified. Among detected compounds, several phenolic acids, phenolic glycosides, and organic acids were found. Within this diversity, 26 metabolites were exclusively detected in the aerial part, and 19 in the roots. Twenty-four metabolites occurred in both plant parts. According to the relative abundance of peaks in the chromatogram, ferulic and piscidic acids and their derivatives may correspond to one of the main phenolic compounds of C. macromeris. Our results contribute to the phytochemical knowledge regarding C. macromeris and its potential applications in the pharmaceutical and cosmetic industries. Besides, some metabolites and their fragmentation patterns are reported here for the first time for cacti species.Chromatographic separation combined with mass spectrometry is a powerful tool for the characterization of plant metabolites because of its high sensitivity and selectivity. In this work, the phytochemical profile of aerial and radicular parts of  The family Cactaceae is one of the most threatened within the plant kingdom , and is Plants contain different classes of metabolites found in different concentrations and of a wide structural diversity. Their phytochemical profiles and functional properties may vary according to: a) the solvent and extraction processes ,15, b) tCoryphantha is highly endemic, distributed in the arid and semiarid regions located between the Sierra Madre Oriental and the Sierra Madre Occidental in northern Mexico, expanding to the southern regions of Arizona, Texas, and New Mexico, USA  was: , , , , , , , and 12 min for column equilibration before each injection. The system was controlled by Chromeleon 7.2 Software  and hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo Fisher Scientific). The chromatographic system was coupled to the mass spectrometer with a heated electrospray ionization source II (HESI II). Nitrogen (purity > 99.999%) was employed as both the collision and damping gas. Nitrogen was obtained from a Genius NM32LA nitrogen generator . Mass calibration for Orbitrap was performed once a week, in both negative and positive modes. Caffeine and N-butylamine  were the calibration standards for positive ions and buspirone hydrochloride, sodium dodecyl sulfate, and taurocholic acid sodium salt were used to calibrate the mass spectrometer. These compounds were dissolved in a mixture of acetic acid, acetonitrile, water, and methanol  and infused using a Chemyx Fusion 100 syringe pump. XCalibur 2.3 software and Trace Finder 3.2  were used for UHPLC control and data processing, respectively. Q Exactive 2.0 SP 2  was used to control the mass spectrometer. Phytochemical analysis was performed as reported previously ,33,34, um/z 200. For the compounds of interest, a scan range of m/z 100–1000 was chosen; the automatic gain control (AGC) was set at 3 × 106 and the injection time was set to 200 ms. Scan rate was set at 2 scans s−1. External calibration was performed using a calibration solution in positive and negative modes before each sample series. In addition to the full scan acquisition method, for confirmation purposes, a targeted MS/MS analysis was performed using the mass inclusion list and expected retention times of the target analytes, with a 30 s time window, with the Orbitrap spectrometer operating both in positive and negative mode at 17,500 FWHM (m/z 200). The AGC target was set to 2 × 105, with the maximum injection time of 20 ms. The precursor ions were filtered by the quadrupole operating at an isolation window of m/z 2. The fore vacuum, high vacuum, and ultra-high vacuum were maintained at approximately 2 mbar, from 105 to below 1010 mbar, respectively. Collision energy (HCD cell) was operated at 30 eV. Detection was based on calculated exact mass and on retention time of target compounds presented in The HESI parameters were optimized as follows: sheath gas flow rate 75 units; auxiliary gas flow rate 20 units; capillary temperature 400 °C; auxiliary gas heater temperature 500 °C; spray voltage 2500 V (for ESI-); and S lens RF level 30. Full scan data in negative mode was acquired at a resolving power of 70,000 full width half maximum (FWHM) at C. macromeris, and the identification of most of them. The aerial part showed higher diversity of metabolites compared with roots, and 24 metabolites occurred in both plant parts. Among detected compounds, ferulic and piscidic acid and their derivatives were the most recurrent phenolic metabolites in C. macromeris. In addition, different classes of compounds with reported functional properties were detected. To our knowledge, the phytochemical profile of aerial and root parts of C. macromeris is reported here for the first time by means of modern ultra-high-performance liquid chromatography–tandem mass spectrometry. Based on the detected compounds, C. macromeris may find potential applications in the pharmaceutical and cosmetic industries. The obtained information could be also useful as chemotaxonomic markers for Coryphantha species.The utilized procedures allowed the separation of 69 metabolites present in methanolic extracts of aerial and radicular parts of


Cigarette smoking is associated with earlier menopause, but the impact of being a former smoker and any dose-response relationships on the degree of smoking and age at menopause have been less clear. If the toxic impact of cigarette smoking on ovarian function is irreversible, we hypothesized that even former smokers might experience earlier menopause, and variations in intensity, duration, cumulative dose, and age at start/quit of smoking might have varying impacts on the risk of experiencing earlier menopause.A total of 207,231 and 27,580 postmenopausal women were included in the cross-sectional and prospective analyses, respectively. They were from 17 studies in 7 countries  that contributed data to the International collaboration for a Life course Approach to reproductive health and Chronic disease Events (InterLACE). Information on smoking status, cigarettes smoked per day (intensity), smoking duration, pack-years (cumulative dose), age started, and years since quitting smoking was collected at baseline. We used multinomial logistic regression models to estimate multivariable relative risk ratios (RRRs) and 95% confidence intervals (CIs) for the associations between each smoking measure and categorised age at menopause (<40 (premature), 40–44 (early), 45–49, 50–51 (reference), and ≥52 years). The association with current and former smokers was analysed separately. Sensitivity analyses and two-step meta-analyses were also conducted to test the results. The Bayesian information criterion (BIC) was used to compare the fit of the models of smoking measures.p < 0.001) and early menopause  (p < 0.001). The corresponding RRRs in former smokers were attenuated to 1.13  and 1.15 . In both current and former smokers, dose-response relationships were observed, i.e., higher intensity, longer duration, higher cumulative dose, earlier age at start smoking, and shorter time since quitting smoking were significantly associated with higher risk of premature and early menopause, as well as earlier menopause at 45–49 years. Duration of smoking was a strong predictor of age at natural menopause. Among current smokers with duration of 15–20 years, the risk was markedly higher for premature  and early  menopause. Also, current smokers with 11–15 pack-years had over 4-fold  and 3-fold  risk of premature and early menopause, respectively. Smokers who had quit smoking for more than 10 years had similar risk as never smokers . A limitation of the study is the measurement errors that may have arisen due to recall bias.Overall, 1.9% and 7.3% of women experienced premature and early menopause, respectively. Compared with never smokers, current smokers had around twice the risk of experiencing premature   and age at natural menopause have been less clear.We pooled individual-level data from 17 observational studies that contributed to the International collaboration for a Life course Approach to reproductive health and Chronic disease Events (InterLACE) consortium. A total of 207,231 and 27,580 postmenopausal women were included in the cross-sectional and prospective analyses, respectively.We examined the associations between smoking measures  and age at natural menopause.Compared with never smokers, former smokers had around 15% higher risk of premature and early menopause, but this was much lower than that of current smokers, who had double the risk when compared to never smokers.Early menopause is positively associated with intensity, duration, cumulative dose, and earlier initiation of smoking in both current and former smokers. Duration of smoking was a strong predictor of early menopause.Our findings highlight the clear benefits for women of early smoking cessation to lower their excess risk of earlier menopause, thereby reducing the increased risks of chronic diseases  associated with both smoking and earlier menopause. Natural menopause is defined as when a woman has had no menstrual periods for 12 consecutive months that did not result from interventions . It typically occurs between 49 and 52 years of age, with a median age of 51.4 years in high-income countries . ApproxiIn addition to genetic factors , socioecTo investigate these issues, we analysed individual-level data from more than 200,000 postmenopausal women in 17 studies that contributed data to the International collaboration for a Life course Approach to reproductive health and Chronic disease Events (InterLACE) ,23. DoseInterLACE has pooled individual-level data on reproductive health and chronic diseases from over 500,000 women from 25 observational studies across 10 countries. Most studies are prospective and collected survey data on key reproductive, sociodemographic, and lifestyle factors, as well as disease outcomes. Participants in each of the included studies were recruited and provided consent according to the approved protocols of the Institutional Review Board or Human Research Ethics Committee at each relevant institution. More detailed descriptions of the InterLACE consortium have been published previously ,23. For Age at natural menopause was the outcome variable and was self-reported. In the cross-sectional analyses, age at menopause was categorised as <40 years (premature menopause), 40–44 (early menopause), 45–49, 50–51 (reference category), and 52 years and above, while in the prospective analyses—due to the insufficient number of women with premature menopause—age at menopause was classified into 4 categories by combining the premature and early menopause groups as <45 years.Information on smoking status, intensity, duration, and timing of cigarette smoking was collected through self-reported questionnaires. Smoking status was categorised as current, former, or never. Current smokers were defined as women who started smoking before menopause and were regular smokers at baseline or who experienced menopause before they quit smoking. Former smokers were defined as women who experienced menopause after they quit smoking at least 1 year. Smoking intensity was defined as the average number of cigarettes smoked per day categorised as 1–9, 10–19, and 20 cigarettes or more. Among former smokers, duration was defined as the difference between the reported age at starting and quitting and, for current smokers, the difference between the age at starting and age at menopause. Therefore, by definition of duration in current smokers, women with later menopause would tend to have a longer duration. In order to observe the association between smoking duration and earlier menopause, we focused on short duration of smoking (similar reason for categories of pack-years and years since quitting smoking). To determine appropriate categories for duration of smoking, we used a fractional-polynomial model to plot the shape of the relationship between smoking duration  and age at menopause. As expected, we found a U-shape, and duration of 20 years was determined as an appropriate cut-point to define a maximum duration that had an impact on age at menopause. Duration of smoking was then categorised as <10, 10–14, and 15–20 years. The cumulative dose of smoking was calculated as pack-years (the number of cigarettes smoked per day divided by 20 [assuming 20 cigarettes a pack] and multiplied by the duration of smoking) . We cate2, 18.5 to 24.9 kg/m2, 25 to 29.9 kg/m2, and ≥30 kg/m2. Years of education was categorised into ≤10, 11–12, and >12 years. Race/ethnicity/region was combined into one, with 7 categories: White European, White Australian/New Zealand, White American/Canadian, Japanese, Other Asian, African American/Black, and Other. Parity was categorised as none, one child, two children, and three or more children. Age at menarche was divided into 5 categories as ≤11, 12, 13, 14, and 15 years or more.Baseline BMI, years of education, race/ethnicity/region, parity, and age at menarche were used as covariates because these have been shown to be associated with age at menopause ,15. BMI The data analyses for the present study were performed following a prospective research proposal . We perfBecause the UK Biobank data contributed more than 60% of the total sample used in the cross-sectional analyses, we conducted a sensitivity analysis by excluding this study. Also, we analysed the associations of intensity, duration, age of starting smoking, and years since quitting with age at menopause by adjusting for all covariates, including the other smoking characteristics. Finally, in order to evaluate the heterogeneity among studies, in both cross-sectional and prospective analyses, we also performed study-specific regressions and random-effects meta-analysis for the associations between each level of the cigarette smoking measures and each category of menopausal age to estimate the magnitude of association.Statistical analyses were performed by using SAS  and Stata . In SAS, the SURVEYLOGISTIC procedure was used with the generalized logit link to adjust for the clustering of data within studies to obtain robust standard errors. The SGPANEL procedure was used to plot the associations. In STATA, the METAN command was used to perform meta-analysis. All statistical tests were based on the two-sided 5% level of significance.The mean age at baseline was 58.0 years (range 42.0 to 74.5 years), with more than half of the women born between 1940 and 1949 . Most wop < 0.001) and early  menopause as well as earlier menopause at 45–49 years . In former smokers, the corresponding RRRs were substantially attenuated to 1.13 , 1.15 , and 1.09 , respectively (p < 0.001) and 6.55 , respectively, in duration 15–20 years. In both current and former smokers, dose-response relationships were observed in all measures of smoking characteristics, i.e., higher intensity, longer duration, higher cumulative dose, earlier age at start smoking, and shorter time since quitting smoking were significantly associated with higher risk of premature and early menopause, as well as earlier menopause at 45–49 years (p < 0.001) and early  menopause, which were close to the levels of risk seen in current smokers  and early  menopause were no longer significant (p = 0.061) compared with the reference group (women who had quit smoking for at least 11 years). The study-specific regressions and random-effects meta-analysis for the associations between each level of cigarettes smoking exposures and each category of menopause showed consistent results with the main analysis  at a subsequent survey, some estimates of association for early menopause were attenuated  or with wide CIs  . The stuOur results indicate that compared with never smokers, both current smokers and former smokers are at higher risk of premature and early menopause and menopause at 45–49 years, although the association is weaker among former smokers. Intensity, duration, cumulative dose, younger age at start smoking, and shorter time since quitting smoking are all associated with earlier age at menopause in both current and former smokers, but the excess risks were substantially lower in former smokers. Consistent dose-response relationships were observed in both current and former smokers. Smoking duration is a much stronger predictor of earlier menopause than others. Current smokers with long duration (15–20 years) showed particularly higher risk of premature and early menopause. These findings highlight the clear benefits of early smoking cessation to lower the excess risk of earlier menopause.Current smoking is an established risk factor for earlier menopause ,15,16, wSeveral hypotheses might explain why smoking leads to earlier menopause. First, cigarette smoke yields polycyclic aromatic hydrocarbons (PAHs), which activate the aromatic hydrocarbon receptor. This receptor induces expression of the apoptosis-promoting gene Bcl2-associated X protein (BAX) in oocytes, which increases the rate of oocyte apoptosis leading to earlier ovarian failure . This meSome studies have reported a potential dose-response relationship between intensity of smoking and earlier menopause ,18,28, wEvidence of the association between duration of smoking and age at menopause has been inconsistent ,18,26. ANumber of pack-years reflects a person’s cumulative cigarette exposure over time. Of the four cross-sectional studies that examined the association between pack-years and age at menopause ,18,30,38Some studies found a dose-response relationship with younger age at starting to smoke and earlier age at menopause ,18, whilSome limitations need to be considered when interpreting these results. Current smoking status was determined at the baseline survey. For women who experienced menopause after baseline, their smoking status was measured at varying years before menopause. Thus, the possibility of measurement errors in smoking status may have arisen. Smoking, however, is a habit that is resistant to change. In studies including women who reported smoking status both before and after menopause , the concordance was over 83%, supporting the use of smoking exposure at baseline as a proxy for smoking status close to or after menopause. For example, when van Asselt and colleagues  repeatedThe main strength of this study was the use of pooled individual-level data from 17 studies across different geographic regions and populations, which resulted in sufficient statistical power to perform both cross-sectional and prospective analyses in current and former smokers separately. The percentages of women with missing data on smoking status, BMI, education level, and race/ethnicity were relatively small (ranging from 1.3% to 5.4%). To our knowledge, it is the first study of this size to examine and quantify the dose-response relationships between intensity, duration, cumulative dose, timing of smoking, and age at menopause separately for former and current smokers. Also, the participant-level data in InterLACE enabled harmonizing variables using common definitions, coding and cut points, which is not usually possible with meta-analyses of published results.Our pooled analysis provides robust evidence that both former and current smokers are at an increased risk of experiencing earlier menopause, but the risk is considerably lower for former smokers. We found significant relationships between the intensity, duration, cumulative dose, the timing of starting, and years since quitting smoking with the risk of earlier menopause in both current and former smokers. Duration and cumulative dose of smoking are good predictors of age at menopause. Current smokers with 15 or more years of smoking duration experience a 15-fold increased risk of premature and a 6-fold increased risk of early menopause. Given that 175 million women ages 15 or older are current smokers globally , our finS1 Text(DOC)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


A battery of toxicological studies was conducted in accordance with international guidelines to investigate the genotoxicity and repeated-dose oral toxicity in rats of synthetic curcumin . There was no evidence of mutagenicity in a bacterial reverse mutation test, whereas an in vitro mammalian chromosomal aberration test was positive for induction of chromosomal aberrations which is in line with results reported for natural curcumin. There was no evidence of genotoxicity in an in vivo mammalian micronucleus test. Synthetic curcumin did not cause mortality or toxic effects in a 90-day repeated-dose oral toxicity study at daily doses of 250, 500, or 1000 mg/kg body weight (bw)/day (administered by gavage in a split dose). The no observed adverse effect level (NOAEL) determined from the 90-day study was 1000 mg/kg bw/day for both male and female Wistar rats. Curcuma longa  [Curcumin, a diarylheptanoid, is the most abundant of several natural curcuminoids found in the rhizomes ofurmeric) , 2. Turmurmeric) ; recentlurmeric) .μg/mL of curcumin had no mutagenic effect; however, curcumin at higher concentrations (10–40 μg/mL) appeared to induce mitochondrial and nuclear DNA damage in a dose-dependent manner [Toxicological studies performed on various forms  and amounts of curcumin and other curcuminoids as mixtures have yielded varied results. Some studies in mice have shown signs of hepatotoxicity while others show no or equivocal toxicologically relevant effects ; similart manner . In humat manner , 14.We present herein a battery of Organization for Economic Cooperation and Development (OECD) compliant, in vitro, and in vivo toxicological studies on a novel bioidentical synthetic curcumin (VEAMIN 99) of >99% purity  as a contribution to the scientific knowledge of this individual compound.All chemical reagents, solvents, pharmaceuticals, and other chemicals used in the studies were of analytical or pharmaceutical grade.The test article is a synthetic curcumin, a bright yellow to orange solid compound manufactured by Laurus Labs . The sponsor provided curcumin of 99.4% purity from batch 25027-1VSP10410915, manufactured in August of 2015. Based on the results of preliminary solubility and compatibility tests with bacterial strains used in the bacterial reverse mutation test and human peripheral blood lymphocytes (HPBLs) used in the in vitro chromosomal aberration test, DMSO was chosen as the test article vehicle and vehicle control. Due to the physical characteristics of the test article, the vehicle and vehicle control for the in vivo mouse micronucleus test and 90-day repeated-dose oral toxicity test were 0.5% w/v carboxymethylcellulose sodium salt . Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 and S9 metabolic activation system (S9) were purchased from Molecular Toxicology, Inc. .A bacterial reverse mutation test was conducted to investigate the mutagenic potential of synthetic curcumin according to the procedures described by Ames et al. , Green aFor the preliminary cytotoxicity assay, test solutions were prepared by dissolving curcumin in DMSO to achieve concentrations of 16.0, 5.0, 1.6, 0.5, 0.16, 0.05, and 0.016 mg/mL. For the mutagenicity assay, test solutions were prepared by dissolving the test item in DMSO to achieve concentrations of 5.0, 1.6, 0.5, 0.16, and 0.05 mg/mL. All test item preparations and dilutions were carried out under sterile conditions. S9 mix  and positive controls were prepared freshly on the day of the experiment. Sodium azide and mitomycin C were diluted in water; all other positive controls were diluted in DMSO.μg/plate. Positive controls for the experiments without S9 were 2-nitroflourine (25.0 μg/plate) for TA98, sodium azide (20.0 μg/plate) for TA100 and TA1535, and 9-aminoacridine (50.0 μg/plate) for TA 1537 and mitomycin C/ametycine (0.25 μg/plate) for TA102. The positive control for all of the experiments with S9 was 2-aminoanthracine (20.0 μg/plate).A preliminary cytotoxicity assay was performed utilizing the plate incorporation method in triplicate by exposing tester strains TA98 and TA100 with and without metabolic activation to the following concentrations of the test article: 1.6, 5.0, 16.0, 50.0, 160.0, 500.0, 1600.0, and 5000.0 μg/plate. All treated plates were incubated at 37 ± 2°C for 68:25 (hours:minutes) in the preliminary cytotoxicity test and 66:10 (hours:minutes) in the mutagenicity assay after which the plates were manually examined for background lawn inhibition, precipitation, and revertant colonies.The mutagenic assay was performed utilizing the plate incorporation method, in triplicate, by exposing tester strains TA98, TA100, TA102, TA1535, and TA1537, with and without S9, to the following test article concentrations: 5.0, 16.0, 50.0, 160.0, 500, and 1600.0 there was at least a 2-fold increase  or 3-fold increase (for TA1535 and TA1537) in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control;the increase in the mean number of revertants per plate was accompanied by a dose response in a minimum of 2–3 concentrations.A result was considered positive ifThe in vitro mammalian chromosomal aberration test was conducted to evaluate the ability of curcumin and/or its metabolites to induce structural chromosome aberrations in cultured HPBL. It was performed in compliance with OECD 473  and GLP Test article formulations were prepared on the day of treatment by diluting stock solution with DMSO to achieve the test concentrations. HPBLs were obtained by drawing blood from healthy, young, nonsmoking males with no known illness or recent exposure to genotoxic agents and subsequently pooling and culturing blood in Roswell Park Memorial Institute Medium, with 15% Fetal Bovine Serum (FBS). Whole blood cultures were incubated at 37 ± 2°C in a humidified environment.μg/mL for experiments without metabolic activation, and cyclophosphamide, dissolved in water to a concentration of 12.5 μg/mL for experiments with metabolic activation.Positive controls were mitomycin C/ametycine, dissolved in water to a concentration of 0.25 μg/mL for four hours; additional HPBL cultures were continuously exposed to the same concentrations without metabolic activation for 22 hours. Experiments for all test groups including the vehicle control were performed in duplicate. At least one thousand cells in each culture were analyzed for mitotic index . Cytotoxicity was defined as a reduction in MI to 45 ± 5% of the vehicle control.A preliminary cytotoxicity assay was performed to determine the test concentrations for the chromosome aberration assay. HPBL cultures were exposed to the test article with and without metabolic activation at concentrations of 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, and 250.0 μg/mL in the absence of metabolic activation, and to concentrations of 6.3, 12.5, and 25.0 μg/mL in the presence of S9 metabolic activation and to corresponding positive and negative controls, and incubated. Following incubation, all cultures were washed with plain media and placed into fresh culture medium with 15% FBS to continue incubation until harvest.The chromosome aberration assay consisted of two independent, concurrent experiments, a short-term exposure assay and a continuous exposure assay. In the short-term exposure assay, cells were exposed to the test article at concentrations of 10.0, 20.0, and 40.0 μg/mL, vehicle, and positive controls, and incubated in the absence of metabolic activation. Culture media was changed at the time of cell harvest. The pH was measured before and after all experimentsIn the continuous exposure experiment, cells were exposed to the test article at concentrations of 6.3, 12.5, and 25.0 Approximately 20 hours after exposure initiation in all experiments, 0.1 mL of colchicine was added to arrest mitosis. Approximately 2.5 hours after application of colchicine  cells were harvested and chromosome slides were prepared for analysis.Slides were coded and scored blind and at least 1000 cells from each group were evaluated for MI. Scoring occurred on the basis of good chromosome morphology and only cells with equal numbers of centromeres and modal numbers (46 ± 2) were analyzed. Three hundred metaphases (150 from each duplicate) were evaluated for structural chromosome aberrations. The percent of polyploidy and endoreduplication was calculated by evaluating 250 metaphases per culture. Gaps were recorded separately but were not included in the total aberration frequency as gaps are considered achromatic lesions similar to nucleolar constrictions, which are easily broken by the pressure exerted during slide preparation  or most The test was considered positive if a significant increase in the number of cells with chromosome aberrations was observed at one or more test concentrations and the increase was dose-dependent. The test was considered negative if none of the above criteria were met under all experimental conditions.The Institutional Animal Ethics Committee (IAEC) of Vimta Labs approved the in vivo mouse micronucleus test and 90-day repeated oral dose study protocols. The ethical practices set forth by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) were followed throughout studies.The in vivo mouse micronucleus test was conducted to evaluate the genotoxic potential of synthetic curcumin to induce the formation of micronuclei in polychromatic erythrocytes (PCEs) in the bone marrow of mice. The study was conducted in compliance with OECD 474 2014)  and GLP 14  and GOn the day of treatment, the test article was suspended in 0.5% w/v carboxymethylcellulose (CMC) sodium salt in water  under sterile conditions to achieve concentrations of 25, 50, and 100 mg/mL and was administered at a dose volume of 10 mL/kg body weight (bw). Cyclophosphamide served as the positive control and was prepared on the day of treatment by dissolving 30.09 mg of cyclophosphamide monohydrate in deionized water to a concentration of 3 mg/mL. 0.5% w/v CMC sodium salt solution served as the vehicle control. ad libitum.Seven-to-nine-week-old male and female Swiss Albino mice weighing 22.27–33.22 grams were utilized for this study. The mice were acclimatized, observed, and examined for a period of 5 days, per Vimta Labs' standard operating procedures, to confirm that the animals were in good health. Animals were housed in groups of up to three in sterilized suspended polycarbonate cages with laboratory bedding material; the room temperature was 21.1–22.9°C with 41.0–62.0% relative humidity and 12-hour light-dark cycles. All animals had access to standard pellets (Harlan Laboratories) and waterAnimals were randomized into groups of 5 animals/sex/group at dose levels of 500, 1000, and 2000 mg/kg bw/day and negative and positive control groups. The test article and vehicle control were administered for two consecutive days, 24 hours apart, in divided doses (about 1–2 hours apart) by oral gavage with the dose volume maintained at 20 mL/kg bw. The positive control was administered 24 hours prior to sacrifice in a single dose of 30 mg/kg bw by gavage with dose volume of 10 mL/kg bw. Animals were observed for clinical signs at the time of dosing, one hour after treatment, and once daily thereafter, while being observed twice daily for mortality or moribund condition. Body weights were recorded on the day of receipt, before randomization, on treatment days, and the day of sacrifice. Bone marrow samples were collected from both exposed femurs of each animal 24 hours after the last dose. Three slides per animal were prepared, blind coded, and examined for incidence of micronucleated cells. A minimum of 4000 PCEs were scored per animal and the frequency of micronucleated PCEs (MNPCEs) was reported as percent of MNPCEs. The proportion of PCE of total erythrocytes (TE) was determined for each animal by counting a total of at least 500 erythrocytes.At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control.This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test.Criteria for a positive result were as follows:The 90-day study was conducted to evaluate the potential toxicity profile and target organs of repeated exposure to synthetic curcumin in male and female Wistar rats and to determine the no observed adverse effect level (NOAEL). The study was conducted in compliance with OECD 408 (1998)  and OECDThe test article was suspended in 0.5% w/v CMC sodium salt solution in water  to achieve concentrations of 12.5, 25, and 50 mg/mL. The reference item, a natural curcumin powder containing 97.9% w/w total Curcuminoids of Curcumin (80.1%), demethoxycurcumin (15.4%), and bisdemethoxycurcumin (2.4%) , was included for comparison of toxicological profiles between the curcumin test article and a natural curcumin with a different purity level under the same test conditions and was prepared in the same manner as the test article. All test article formulations were analyzed by high-performance liquid chromatography in the first, seventh, and last weeks of the treatment period and all results fell within the qualifying limit of 85–115% of curcumin. Formulation stability was tested and found to be stable for 48 hours at room temperature. ad libitum except during fasting days when they received water only. Throughout acclimatization and treatment periods, animal rooms were maintained at 20.8–23.7°C and 38–68% relative humidity with light/dark cycles of 12 hours.Following a detailed clinical exam on the fifth day of acclimatization, Wistar rats , 5–7 weeks old, and weighing 170.83 ± 12.95 g  and 137.14 ± 10.37 g  were randomly divided by zigzag manual method based on body weight, into 5 groups of 20 rats/sex/group . Animals were housed in same-sex pairs in sterilized polycarbonate cages in which they had access to reverse osmosis filtered water and certified standard pelleted laboratory animal diet Doses and the divided-dose (bid) administration regimen were chosen based on solubility trials (single-dose suspensions were too viscous to administer) and a 14-day repeated-dose oral toxicity, non-OECD/GLP compliant dose range-finding study on Wistar rats . There were no toxic changes in the assessed parameters of the 14-day study and no test article related changes other than stool color. Therefore, it was concluded that the NOAEL for the test article after 14-day repeated-dose administration was 2000 mg/kg bw/day (1000 mg/kg bw bid), the highest dose tested. Although there were dose-dependent increases in absolute and relative liver weights in males of the 1000 and 2000 mg/kg bw/day groups, these findings were not considered toxicologically relevant due to lack of correlating clinical chemistry, gross pathological, or histopathological findings. Due to concern of greater organ weight increases with longer-term treatment, the following doses were chosen for the 90-day repeated-dose oral study: 250 mg/kg bw/day (125 mg/kg bw bid), 500 mg/kg bw/day (250 mg/kg bw bid), and 1000 mg/kg bw/day (500 mg/kg bw bid). The reference group received 1000 mg/kg bw/d (500 mg/kg bw bid) and the vehicle control received an equivalent volume of vehicle formulation, 0.5% CMC in sterile water. Each dose was administered by gavage, at least five hours apart, at a volume of 10 mL/kg bw, based on the most recent recorded weight of the animals.Animals were observed for morbidity and mortality twice daily. General clinical observations occurred daily, and detailed clinical examinations occurred before randomization and weekly thereafter throughout the treatment period. Body weights for all groups were measured prior to gavage on day 1 and weekly until and on the day of sacrifice. Functional observations of all animals in the study took place during week 13 and consisted of observations of the animals in their home cage , during handling , in the open field , and for neuromotor activity .Cage-wise food consumption was recorded weekly and calculated as the difference between food offered (grams) and food left over (grams) divided by the number of rats in the cage. Ophthalmologic examination was performed on all animals prior to the treatment period and on high-dose and control groups during the last week of the study.2 asphyxiation) and underwent gross pathological examination after which absolute and relative organ weights were determined. Histopathological examination was performed on all preserved tissues of high-dose test and reference groups and vehicle control animals. Lung tissue of all low-dose and middose animals was examined following observations of histopathological findings in lungs of the high-dose group.After termination of treatment, three fasting blood samples from each animal were collected from the retroorbital plexus under isoflurane anesthesia for measurement of hematology, clinical chemistry, and coagulation parameters. Urine was collected at the end of the treatment period on day 91 for macroscopic and microscopic examination. On day 91, all animals were weighed and sacrificed . Per the test guidelines, the bacterial reverse mutation test results were interpreted based on the criterion of statistically significant changes; thus, no further statistical analysis was conducted. For the in vitro mammalian chromosomal aberration test, the Cochran-Armitage test for linear trend and Fisher's Exact Test were used to compare the percentage of cells with aberrations in treated cells to the results for the vehicle control. The number of aberrations in the treatment and positive control groups were compared to the corresponding negative control, and all groups were compared to laboratory historical data. For the mouse micronucleus test, the Cochran-Armitage test was used for linear trend and Fisher's Exact Test was used to compare the frequency of MNPCEs among the TEs in the treatment groups to the vehicle controls. Kruskal-Wallis Nonparametric Analysis of Variance (ANOVA) test was used to compare the ratio of PCEs to TE (PCE/TE) of the vehicle control group to the treatment groups. In the 90-day repeated-dose oral toxicity study, the D'Agastino and Pearson Omnibus tests were used to confirm the normality of the data. Normal data were tested with Levene's test for homogeneity of variance. Nonhomogenous data were appropriately transformed before analysis. Student'sμg/plate. Evaluation for mutagenicity revealed no 2-fold or greater increases for TA100, TA102, or TA98 and no 3-fold or greater increases for TA1535 or TA1537 in mean revertants per plate. Also, there were no dose-related increases in the mean number of revertant colonies in any of the tester strains at any test concentration, 5.0–1600.00 μg/plate, with or without S9 compared to vehicle control , no treatment related changes were seen in urinalysis in test and reference item treated animals.Sporadic statistically significant increase in absolute organ weights of the heart, brain, kidneys, testes, and epididymides were observed in the middose group males. Spleen weights were significantly decreased in reference group males compared to vehicle see . A statiHistopathological examination in males and females revealed several lesions present in individual animals in the control, high-dose, or reference group, while several findings were present at the same frequency or were more frequent in the vehicle control group compared to the high-dose or reference group see .μg/mL treated cells, respectively, compared to vehicle control. The increased frequency in the 25.0 μg/mL treated cells was statistically significant. Based on the positive results criteria for this test, it was concluded that the test article was clastogenic in cultured human peripheral blood lymphocytes under the conditions of this test. The test article did not induce micronuclei in the bone marrow of mice and was considered to be nonmutagenic under the conditions of the performed micronucleus test.In the performed bacterial reverse mutation test, both with and without S9, the test item was considered nonmutagenic. In the in vitro chromosomal aberration test, the test article did not induce structural chromosome aberrations in cultured HPBL in any of the tested concentrations in the short-term and continuous exposure experiments without metabolic activation. However, there was a dose-dependent increase with S9 in the short-term experiment, by 0.34, 1.67, and 4.33% in the frequency of aberrant cells in the 6.3, 12.5, and 25.0 During the 90-day study, there were no mortalities in any of the five groups. The yellow discoloration in the feces and on the fur and tail of the middose, high-dose, and reference animals was attributed to the color and volume of the test article with the yellow color on the tail and fur resulting from external contact with the discolored feces and urine. The discoloration was not considered toxicologically relevant. There were no statistically significant changes in body weights throughout the study and no abnormalities were observed on ophthalmological examination. There were statistically significant changes in feed consumption; however, the changes were sporadic and minimal and did not affect the body weight of the animals. Therefore, the differences observed were not considered toxicologically relevant. There were no abnormalities observed in the functional observations of the animals in their home cages, during handling, in the open field, or with neuromotor activities.Statistically significant changes were observed in hematology parameters among the test groups and reference group males. Significant decreases in hemoglobin and mean corpuscular hemoglobin concentration, increases in red cell distribution width and mean corpuscular volume, changes in mean platelet volume, and decreases in WBCs, monocytes, lymphocytes, eosinophils, and neutrophils showed no dose-dependency and remained within or marginal to historical control ranges; thus, these changes were not considered test article related and were attributed to normal variation. Statistically significant dose-dependent decreases in RBC and monocytes, compared to control, were observed in the male test groups as well as the reference group and the high-dose group mean corpuscular hemoglobin was statistically significantly increased compared to control with apparent dose relation; however, as all of the measures remained within biological range, the changes were considered incidental and within normal variation. Other than statistically significant increases in high-dose males compared to the reference group for WBC and lymphocytes, hematology measures for high-dose and reference group males were similar. Several slight but statistically significant increases and decreases in hematology parameters were observed in the low-dose and middose female groups; as the changes were low in magnitude and there were no significant changes in the high-dose group females (no dose relationship), the changes in the low-dose and middose females were considered incidental and of no toxicological consequence. A statistically significant decrease in reticulocytes was observed in the high-dose females compared to the reference females and was also considered an incidental change with measures remaining within the historical range.Statistically significant differences in clinical chemistry measures in test group males were without dose relation, were present only in the low-dose and/or middose groups, and/or remained within or marginal to historical control ranges; therefore, the changes were considered within the normal variation of the animals and of no toxicological significance. Phosphorus and chloride were statistically significantly increased compared to the reference group with phosphorus which also significantly increased compared to vehicle control with potential dose relation. However, all measures for phosphorus and chloride remained within biological ranges and differences were present without correlating changes in gross pathology or histology.Similarly, clinical chemistry results for female groups revealed statistically significant differences among several measures. However, the changes were not dose-related, remained within or marginal to historical control values, and/or were considered nonadverse ; thus, they were considered within the normal biological range of the animals.Coagulation parameter results revealed statistically significant decreases in prothrombin time (PT) for middose and high-dose males that were marginal to the historical range. Since the decrease in fibrinogen occurred simultaneously with a faster clotting time (PT) and no significant change was seen in platelets, the changes are not considered of toxicological significance. Fibrinogen levels in low-dose males were statistically significantly decreased compared to control with PT, activated partial thromboplastin time (APTT), and platelets remaining unaffected; thus, the change is not considered biologically relevant. The high-dose group result for PT was significantly decreased compared to the reference item and was considered incidental. Additionally, all coagulation results for males fell within or marginal to the historical ranges and were considered within the range of normal variation.Coagulation parameters in females revealed no significant changes in fibrinogen levels and only a statistically significant decrease for the middose group for APTT. PT was statistically significantly increased in the low-dose, middose, and reference groups without dose relationship. The absence of dose relationship, along with no change in platelet counts, the nonadverse change in APTT, and all results falling within historical controls, leads to the conclusion that the changes are not toxicologically relevant.Urinalysis results contained no significant findings. The changes in several absolute organ weights in male groups  showed dose-unrelated, statistically significant increases with no associated changes in gross pathology or histopathology; thus, they were considered incidental findings. In females, liver weight was statistically significantly decreased in the middose group and the adrenal glands were statistically significantly increased in the high-dose group. The changes lacked correlating gross pathological or histopathological findings; thus, the changes were considered incidental.Relative organ weight changes for male kidney weights were not dose-dependent and cooccurred with only one gross kidney lesion each in the low-dose and middose groups. Increases in liver weight relative to body weight in males have apparent dose relationship with nonsignificant increases also seen in the low-dose and middose groups; however, the increases occur in the absence of related gross pathological or clinical chemistry findings and the only hepatic histopathological finding  was in a single high-dose male. Thus, the increases in relative kidney and liver weights are not considered toxicologically relevant. Since the only statistically significant changes in organ weight ratios for females were increases in the lungs and spleen of the low-dose group, the findings were considered incidental.The yellow discoloration of the lungs in test article group males and females and in reference groups, along with the histopathological findings , was consistent with findings associated with oral gavage error, spontaneous lesions, and/or aspiration of the test and reference solutions , 27. In μg/mL or more [In the studies reported herein, the test article, synthetic curcumin, was found to be nonmutagenic in the bacterial reverse mutation test, positive for clastogenic activity in the in vitro chromosomal aberration test, and nongenotoxic in the mammalian micronucleus test, with a NOAEL of 1000 mg/kg bw/day in this 90-day repeated-dose oral toxicity study. Natural curcumin has been shown to induce chromosomal aberrations in cells at various stages of cell division at levels of 5–10  or more –36. Inve or more , 37, 38. or more , 33. In  or more . In conc


Emotional information significantly affects cognitive processes, as proved by research in the past decades. Recently, emotional effects on language comprehension and, particularly, syntactic processing, have been reported. However, more research is needed, as this is yet very scarce. The present paper focuses on the effects of emotion-laden linguistic material (words) on subsequent morphosyntactic processing, by using Event-Related brain Potentials (ERP). The main aim of this paper is to clarify whether the effects previously reported remain when positive, negative and neutral stimuli are equated in arousal levels and whether they remain long-lasting. In addition, we aimed at testing whether these effects vary as a function of the task performed with the emotion-laden words, to assess their robustness across variations in attention and cognitive load during the processing of the emotional words. In this regard, two different tasks were performed: a reading aloud (RA) task, where participants simply read aloud the words, written in black on white background, and an Emotional Stroop (ES) task, where participants named the colors in which the emotional words were shown. After these words, neutral sentences followed, that had to be evaluated for grammaticality while recording ERPs . ERP analyses showed main effects of valence across tasks on the two components reflecting morphosyntactic processing: The Left Anterior Negativity (LAN) is increased by previous emotional words (more by negative than positive) relative to neutral ones, while the P600 is similarly decreased. No interactions between task and valence were found. As a result, an emotion-laden word preceding a sentence can modulate the syntactic processing of the latter, independently of the arousal and processing conditions of the emotional word. Contrary to previous traditions in the cognitive sciences, emotions seem to strongly impact cognitive processes. Numerous experiments have demonstrated how positive or negative emotions affect processes such as perception, attention, memory, creativity and other higher cognitive functions . In this regard, emotional information has been able to modulate at least two ERP components normally related to morphosyntactic processing. One of these components is the Left Anterior Negativity (LAN), triggered by syntactic anomalies . LAN is Some studies have explored the impact of emotional information on morphosyntactic processing when emotion is enclosed within the sentence to be syntactically analyzed (short-term effects) . OverallValence has not been manipulated independently from arousal in the previous studies aiming to study the impact of emotional information on syntactic processing, yet specific experiments are required in this regard, matching arousal degree between valence conditions, including (importantly) the neutral one. The goal of the present study is to contribute in this respect. The long-term effects of valence on morphosyntactic processing irrespective of arousal are here explored. For the valence manipulation, positive, negative, and neutral words were presented prior to neutral sentences with or without morphosyntactic violations, as did In view of previous findings, meanly those by SD = 8.37) participated in this experiment. All of them were university students and gave informed consent before the experiment. The study was performed in accordance with the Declaration of Helsinki and approved by the ethics committee of the Complutense University, Madrid, Spain. All the participants reported normal or corrected-to-normal vision and no history of neural or cognitive disorders or reading difficulty. All were right-handed, ranging from 30 to 100 (M = 83) according to the Edinburgh Handedness Inventory (SD = 9.8) and a trait-anxiety mean of 66.6 (SD = 15.3).Twenty-four native Spanish speakers -(noun)-(adjective)-(verb)] was like other sentences already used in previous experiments . Half ofSD = 0.73) and arousal mean was 4.7 (SD = 0.8) in a 1–9 scale. Accordingly, they could be considered of neutral valence and medium arousal. For each sentence, two additional ones were generated either containing a gender disagreement or a number disagreement between noun and adjective. That is, we had 360 correct sentences, 360 sentences with a gender disagreement in the adjective and 360 sentences with a number disagreement in the adjective. Examples of the sentences can be found in Table Our critical word (CW) in the sentences was the adjective. We selected neutral adjectives from the Spanish adaptation of the ANEW . AdjectiTo counterbalance stimuli presentation, the complete scenario of 360 sentences had to be presented to each participant in six sets of 60 sentences each. Every set was constructed with 30 correct and 30 incorrect sentences . Within every set, the 60 sentences were randomly mixed during the presentation. A total of 12 different combinations of 6 sets each were constructed, so that each participant viewed every one of the 360 sentences only once and in only one version (correct or one of the two incorrect versions). Across the 12 combinations, all the sentences appear counterbalanced across versions, in different participants. Since we had 12 combinations and 24 participants, each combination was presented twice.SD = 0.36), for negative words was 2.29 (SD = 0.48), and for neutral ones was 5.01 (SD = 0.56), in a 1–9 scale. Arousal levels were carefully matched across conditions to such an extent that the mean arousal value for positive, negative and neutral condition was the same, 5.03, with SD = 0.53, SD = 0.55, and SD = 0.52, for positive, negative, and neutral condition, respectively.In addition, a list of 180 emotional words was constructed, to be presented preceding each experimental sentence. These words were those used in The experiment was block-designed. The use of the emotional words as mood inductors before the sentences resulted in six different conditions: positive, negative and neutral valence, each one both in two tasks —ES and RA. Every block consisted of 60 emotional words , each preceding one of the 60 sentences in a set (see above). All the emotional words in a block had the same valence, to best maintain the induced mood. So, each one of the six blocks had a single combination of valence and task.The RA task consisted in RA the single word presented in black on white prior to the sentence. In the ES task, participants had to inhibit the meaning of the word and read aloud the color in which it was presented. Six different colors were used: red, green, blue, yellow, magenta, and orange. In every block of 60 words, we used 10 times each color, randomizing the order.Participants were comfortably seated in a quiet shielded chamber watching an LCD screen . Each trial began with an asterisk, lasting 500 ms. After an inter-stimulus-interval (ISI) of 300 ms, the emotional stimulus  was presented during 300 ms. Other 300 ms of blank were followed by a fixation cross for 500 ms, indicating the sentence was about to appear. After 700 ms of blank screen, the sentence started, word by word, in black on white, in the center of the monitor, lasting 300 ms each word and with 300 ms of ISI. At the end of the sentence, after 1 s, a question mark was presented for 1 s, lending the subject to respond whether the sentence was correct or incorrect. The first word of each sentence always began with a capital letter; the last word was always presented with an ending dot. The used font was 30-point Arial  used always the same task, that is, either RA or ES. Between blocks, a message of pause appeared; subjects had to press a button to continue. Between the third and the fourth block, preceding task changed . Since the level of arousal was expected to vary between the two parts of the experiment, enough time between the 3rd and the 4th blocks was necessary  to return to the arousal baseline values. The Oldfield test of handedness was filled-in at this moment. Then, when participants were ready, instructions about the new preceding task were given, followed by a training period, before starting with the next –and last- three blocks, with pauses between them. Finally, the participants completed the STAI questionnaire.Skin conductance (SC) was recorded throughout the experiment by means of a Biofeedback (6-channel Psymtec I-330-C2), connected to the index and middle fingers of the hand that was not used to respond. This manner, we gathered information regarding the arousal levels of the participant, to contrast it across conditions. We compared SC means between conditions, after normalizing data within subjects: all individual measures of SCR in μS were averaged for each participant separately. This grand mean was assigned a value of one; thereafter, the specific values as a function of each preceding task were obtained and compared in terms of standard deviations from the grand mean.The electroencephalogram (EEG) was recorded with 59 electrodes attached in a cap  with the locations: Fp1, Fpz, Fp2, AF7, AF3, AF4, AF8, F7, F5, F3, F1, Fz, F2, F4, F6, F8, FT7, FC5, FC3, FC1, FCz, FC2, FC4, FC6, FT8, T7, C5, C3, C1, Cz, C2, C4, C6, T8, TP7, CP5, CP3, CP1, CPz, CP2, CP4, CP6, TP8, P7, P5, P3, P1, Pz, P2, P4, P6, P8, PO7, PO3, PO4, PO8, O1, Oz, and O2, plus the right mastoid (M2). All of them were initially referenced to the left mastoid (M1). For controlling ocular movements, EOG were registered with electrodes above and below the left eye (VEOG), and at the outer canthus of each eye (HEOG), for off-line eye-movement correction. A Brainamp amplifier  was used, keeping electrode impedances below three kΩ. The signal was continuously recorded with a bandpass from 0.01 to 30 Hz and a sampling rate of 250 Hz.The data analysis was performed using Brain Vision Analyzer software . Continuous recording of EEG was divided into epochs of 1000 ms each, starting 200 ms before the onset of the CW. We considered only correct trials, discarding incorrect and out-of- time trials. We corrected eye blinks and EOG artifacts using a semiautomatic Ocular Correction ICA tool (fast-ICA extended); final components rejection was done by visual inspection. Similarly, other artifacts rejection was semiautomatic: an automatic rejection of epochs exceeding ±100 μV was followed by a manual rejection by visual inspection. Data of the 59 locations was referenced again by calculating the mean between M1 and M2 data.post hoc tests were corrected by the Bonferroni procedure. Specific time windows for the analyses were selected based upon visual inspection of the main ERP components.Statistical analyses of variance (ANOVAs) of ERP data were performed using six brain regions of interest (ROIs): anterior, central and posterior regions, each one represented in the two hemispheres Figure . The facSD = 4.7)] than the correct ones [91.8% of cases (SD = 5.2)]. This difference was significant, as supported by a main effect of Correctness  in an ANOVA comprising Correctness, Preceding task and Valence. There were no significant differences regarding other factors or interactions. .Participants appropriately judged the morphosyntactically incorrect sentences more accurately .In line with the error rates findings, reaction times were faster  = 4.78; p = 0.039; η2 = 0.172; and π = 0.554]. As expected, ES task produced a significant increase of SC (standardized M = 1.03) compared to the RA task (standardized M = 0.97). No effects were found on SC concerning valence conditions .The comparison showed a main effect of Preceding task , as well as of a Correctness x Valence interaction . Preceding Task x Region yielded significant effects  while the Preceding Task x Correction x Hemisphere interaction resulted in a trend . All other factors and interactions were not significant . Post hoc analyses revealed that the LAN differed significantly between neutral and negative conditions . No significant differences appeared between neutral and positive conditions  neither between the positive and negative conditions in the LAN . Statistically, negative and positive conditions behave similarly, but also positive and neutral conditions, therefore an intermediate position of positive condition between neutral and negative conditions might be suggested, as a visual inspection of the waves illustrates. Preceding Task effects will not be discussed, because of two main reasons: first, the effects did not interact with Correctness, so they are not affecting the syntactic processes of interest here. Second, there is no straightforward evidence on the specific factor underlying the effect, since different number of them could be implicated .Visual inspections revealed a LAN for the three valence conditions between 350 and 450 ms, with different wave amplitudes, being largest in the negative valence, slightly smaller in the positive, and showing the lowest values in the neutral condition  = 40.339; p < 0.001; η2 = 0.637; and π = 1], and a trend to significance of Correctness x Valence x Region . Since the P600 is distributed mainly in the central and posterior regions, and in order to seemingly reduce statistical power issues, a new ANOVA excluding the anterior region was performed, yielding a main effect of Correctness  and of Correctness x Valence . Post hoc analyses comparing the P600 amplitude for each valence (subtracting the activation of the correct ones to the incorrect ones) revealed a significant difference between negative and neutral conditions , but not between positive and neutral neither between negative and positive . This matches visual inspection of the waves, in which the P600 appeared for the three valences, being larger for the neutral condition, followed by the positive one, the smallest being for the negative one, then reversing the results of the LAN  was also assessed. Although significant differences were found between ES and RA preceding tasks in SC measurement, morphosyntactic processing was not affected by the preceding task type. In contrast, even though SC differences were not observed for valence, emotional valence modulations were observed on the main ERP components related to morphosyntactic processing occurring around 2 s later. Both negative and positive words seemed to enhance the amplitude of the LAN and to reduce the P600 component. Negative valence significantly differed from neutral condition both in the LAN and in the P600 ERP components. There were, however, no significant differences between negative and positive neither between positive and neutral conditions, neither in the LAN nor in the P600 for this comparison. Visual inspection, however, rather suggests an intermediate position of the positive condition between the negative and the neutral ones, although this depiction should be viewed with caution.Similarly to the present experiment, larger LAN amplitudes for negative and positive conditions in comparison to the neutral one were observed in Our results seem to reflect a pattern in which, overall, emotional valences increase LAN amplitude while decreasing P600. This pattern relates to previous reports in which, e.g., individuals with better working memory capacities normally show a larger LAN/reduced P600 in comparison to bad comprehenders or individuals with lower working memory capacity . ApplyinIt is interesting to note that in Valence seems to denote that there is relevant information for the subject . As arousal effects can be entirely disregarded in the present findings, only valence information can uphold. To the extent that the effect was independent common for either type of valence, a general effect must be accounting for our data. It is possible that the semantic content associated to emotional valences triggers an overall strategy by which enhanced processing follows, and this would convey the increase of cognitive resources devoted to process the subsequently appearing sentence. This would in turn result in enhanced LAN/reduced P600, resembling the pattern reported for individuals with larger working memory capacities, as discussed above.Indeed, emotional information is part of the semantic content of a word . TherefoOur results might also be in line with findings in which positive and negative emotions interact differently with attention . In thisIn conclusion, our data suggest that the emotional valence of a word can affect morphosyntactic processing occurring about 2 s later, and that this occurs even when the emotion-laden word has been poorly processed. The result contributes not only to better acknowledge the interactions between emotional-semantic and syntactic processes, but also to recognize that the emotional words that appear in a text, a discourse or in conversations may convey effects on the morphosyntactic processing of sentences appearing far apart.JE, LJ-O, and MM-L designed the study, interpreted the data, discussed the results, and wrote the manuscript. JE and LJ-O collected the data and prepared the data for analysis. All authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.