RESUMEN

Chlormequat (CCC) is widely used in agricultural production to increase the crop yield. However, the effects of CCC on transfer of ARGs in agricultural system are still unclear. In this study, using E.coli DH5α (carrying RP4 plasmid with AmpR, TetR, KanR) as the donor bacterium, E.coli HB101, endophytic Pseudomonas sp. Ph6 or rhizosphere Pseudomonas putida KT2440 as the recipient strain, three conjugative systems were designed to investigate the effects of CCC on ARG transfer. Meanwhile, hydroponics experiments were designed to study the ARG spread in the rice-nutrient solution system after CCC application. The results showed that CCC significantly promoted the RP4 conjugation by expanding cell membrane permeability and improving the relative transcription levels of trfAp, trbBp, traA and traL genes in RP4. Furthermore, the conjugation frequency between E. coli and Pseudomonas was much higher than that between E. coli cells. Compared with spraying foliage with 2500 mg·L-1 of CCC, soaking seeds with 250 mg·L-1 of CCC was more beneficial to the colonization of ARB in rice, and also increased the abundance of ARGs in rice cultivation system. These results remind that the use of CCC in agricultural production might promote the ARG transmission in agro-ecosystems; however, foliage spraying with 2500 mg·L-1 of CCC could control its spread.

Asunto(s)

Conjugación Genética , Escherichia coli , Transferencia de Gen Horizontal , Oryza , Plásmidos , Pseudomonas , Plásmidos/genética , Oryza/microbiología , Oryza/crecimiento & desarrollo , Oryza/efectos de los fármacos , Oryza/genética , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Pseudomonas/genética , Pseudomonas/efectos de los fármacos , Pseudomonas/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/metabolismo , Genes Bacterianos/efectos de los fármacos , Antibacterianos/farmacología , Agricultura , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Microbiana/genética

RESUMEN

Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1ß and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.

Asunto(s)

Proliferación Celular , Ferroptosis , Inflamación , Células Intersticiales del Testículo , Ferroptosis/efectos de los fármacos , Animales , Masculino , Ratones , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Inflamación/patología , Inflamación/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Ciclohexilaminas , Fenilendiaminas

RESUMEN

Chlormequat chloride (CCC), a widely used plant growth regulator, is a choline analogue that has been shown to have endocrine-disrupting effects. Previous studies have shown that maternal exposure to CCC could induce hyperlipidemia and growth disruption in rat offspring. This study aims to further investigate the effects of peripubertal exposure to CCC on pubertal development and lipid homeostasis, as well as the underlying mechanisms. In vivo, male weanling rats were exposed to CCC (0, 20, 75 and 200 mg/kg bw/day) from post-natal day 21-60 via daily oral gavage. The results in rats showed that 75 mg/kg CCC treatment induced hepatic steatosis, predominantly microvesicular steatosis with a small amount of macrovesicular steatosis, in rat livers and 200 mg/kg CCC treatment induced liver damage including inflammatory infiltration, hepatic sinusoidal dilation and necrosis. In vitro, HepG2 cells were treated with CCC (0, 30, 60, 120, 240 and 480 µg/mL) for 24 h. And the results showed that CCC above 120 µg/mL induced an increase in triglyceride and neutral lipid levels of HepG2 cells. Mechanism exploration revealed that CCC treatment promoted the activation of mTOR/SREBP1 signalling pathway and inhibited activation of AMPK in both in vivo rat livers and in vitro HepG2 cells. Treatment with AMPK activator Acadesine (AICAR) could alleviate the lipid accumulation in HepG2 cells induced by CCC. Collectively, the present results indicate that CCC might induce hepatic steatosis by promoting mTOR/SREBP1 mediated lipogenesis via AMPK inhibition.

Asunto(s)

Proteínas Quinasas Activadas por AMP , Clormequat , Hígado Graso , Lipogénesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Serina-Treonina Quinasas TOR , Animales , Serina-Treonina Quinasas TOR/metabolismo , Masculino , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Lipogénesis/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Humanos , Células Hep G2 , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Ratas , Clormequat/toxicidad , Ratas Sprague-Dawley , Hígado/efectos de los fármacos , Hígado/metabolismo

RESUMEN

Since chlormequat chloride is widely applied as a plant growth regulator in agriculture and horticulture, its exposure through food consumption is common. We demonstrated previously that chlormequat chloride exposure during pregnancy led to embryos with bigger sizes associated with higher levels of growth hormone (GH) on gestation day 11 (GD11). However, the dose-effect relationship of chlormequat chloride at a lower dose range was not established, and the underlying mechanisms of its promoting effects on embryonic growth and development were not fully elucidated. To address these, pregnant rats were orally exposed to chlormequat chloride at 0, 0.05, 0.5 and 5 mg/kg.bw from GD0 to 11 and the embryonic growth and growth related hormones were evaluated on GD11. We found that the growth and development of the embryos was significantly promoted in a dose dependent manner by chlormequat chloride. Chlormequat chloride also increased embryonic GH, GH releasing hormone (GHRH), and somatostatin (SRIF), and inhibited the embryonic cAMP dependent protein kinase A (PKA) signaling pathway. Chlormequat chloride increased GH synthesis modulated by GHRH/SRIF-PKA-Pituitary specific transcription factor 1 (Pit-1) in the maternal rats. Intriguingly, chlormequat chloride did not show any effects on GH and PKA signaling pathways in the non-pregnant female rats. These findings together suggest that the disrupting effect of chlormequat chloride on GH is associated with pregnancy.

Asunto(s)

Clormequat , Hormona del Crecimiento , Embarazo , Femenino , Ratas , Animales , Hormona del Crecimiento/metabolismo , Clormequat/toxicidad , Reguladores del Crecimiento de las Plantas/toxicidad , Factores de Transcripción , Transducción de Señal

RESUMEN

Chlormequat chloride (CCC), as a widely used plant growth regulator, can cause impaired sperm quality and decreased testosterone synthesis in pubertal rats, but the underlying mechanism remains unclear. The purpose of this study was to elucidate the toxicokinetics and tissue distribution of CCC, as well as the possible mechanism of CCC-induced impairment in sperm quality. The concentration of CCC reached its peak 1 h after a single dose (200 mg/kg·bw) administration in mice plasma, and a bimodal phenomenon appeared in the testes, liver, and epididymis. In vivo, 200 mg/kg CCC caused testicular damage and impaired sperm quality in pubertal mice, and the expression of p-tyrosine and GSK3α decreased in cauda epididymidis, sperm and testes. CCC also caused the down-regulation of AKAP4 and the up-regulation of calmodulin (CaM), and activated the PI3K/AKT signaling pathway in the testes. In vitro, CCC reduced the levels of p-tyrosine, AKAP4 and GSK3α, increased the level of CaM and activated the PI3K/AKT signaling pathway in GC-1 cells. CaM antagonist (W-7 hydrochloride) and PI3K inhibitor (LY294002) can effectively improve the expression of GSK3α and AKAP4 by suppressing the PI3K/AKT signaling pathway in GC-1 cells treated with CCC. It was indicated that CCC induced impairment in sperm quality might be partially related to the activation of PI3K/AKT signaling pathway mediated by CaM.

Asunto(s)

Acetatos , Clormequat , Fenoles , Proteínas Proto-Oncogénicas c-akt , Ratones , Ratas , Masculino , Animales , Clormequat/metabolismo , Clormequat/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Calmodulina/metabolismo , Calmodulina/farmacología , Semen/metabolismo , Transducción de Señal , Espermatozoides , Tirosina/metabolismo

RESUMEN

Consumers are constantly exposed to a variety of chemical mixtures as part of their everyday activities and lifestyle. Food, water and commercial products are only some examples of the possible ways people get exposed to these mixtures. However, following federal and local guidelines for risk assessment related to chemical exposure, risk analysis focuses on a single substance exposure scenario and not on a mixture, as in real life. Realizing the pronounced gap of this methodology, the real-life risk simulation scenario approach tries to address this problem by investigating the possible effect of long-term exposure to chemical mixtures closely resembling the actual circumstances of modern life. As part of this effort, this study aimed to identify the cumulative effects of pesticides belonging to different classes and commonly used commercial products on long-term exposure with realistic doses. Sprague Dawley rats were given a pesticide mix of active ingredients and formulation chemicals in a daily acceptable dose (ADI) and 10xADI for 90 days. Following thorough everyday documentation of possible side-effects, after 90 days all animals were sacrificed and their organs were examined. Exposure to pesticides particularly affects the miRNA levels at that point will provide us with more information about whether they can be potential biomarkers.

Asunto(s)

MicroARNs , Plaguicidas , Humanos , Ratas , Animales , Plaguicidas/toxicidad , Nivel sin Efectos Adversos Observados , Ratas Sprague-Dawley , Páncreas , Mesenterio

RESUMEN

Presently, the utilization of chlormequat in Astragalus mongholicus Bunge (Leguminosae) cultivation is prevalent for augmenting rhizome (Astragali Radix) yield. However, indiscriminate and excessive chlormequat employment can detrimentally influence Astragali Radix quality and safety. This research aimed to comprehensively comprehend chlormequat risks and its influence on Astragali Radix metabolites. Diverse chlormequat concentrations were employed in Astragalus mongholicus cultivation, with subsequent analysis of residual chlormequat levels in Astragali Radix across treatment groups. Astragali Radix metabolic profiling was conducted through UPLC-QTOF-MS, and thirteen principal active components were quantified via UFLC-MS/MS. Findings revealed a direct correlation between chlormequat residue levels in Astragali Radix and application concentration, with high-dose residue surpassing 5.0 mg/kg. Metabolomics analysis identified twenty-six distinct saponin and flavonoid metabolites. Notably, the application of chlormequat led to the upregulation of seven saponins (e.g., astragaloside I and II) and downregulation of six flavonoids (e.g., methylnissolin-3-O-glucoside and astraisoflavan-7-O-ß-d-glucoside). Quantitative analysis demonstrated variable contents of active ingredients due to differing chlormequat concentrations, leading to astragaloside I increase (14.59-62.55%) and isoastragaloside II increase (4.8-55.63%), while methylnissolin-3-O-glucoside decreased (22.18-41.69%), as did astraisoflavan-7-O-ß-d-glucoside (21.09-47.78%). In conclusion, chlormequat application influenced multiple active components in Astragali Radix, causing constituent proportion variations. Elevated chlormequat concentrations led to increased active components alongside heightened chlormequat residues in Astragali Radix. Consequently, prudent chlormequat application during Astragali Radix production is imperative to avert potential detriments to its quality and safety.

Asunto(s)

Planta del Astrágalo , Medicamentos Herbarios Chinos , Saponinas , Clormequat , Espectrometría de Masas en Tándem , Medicamentos Herbarios Chinos/química , Planta del Astrágalo/química , Astragalus propinquus/química , Flavonoides/análisis , Saponinas/análisis , Glucósidos/análisis

RESUMEN

In this study, surface-enhanced Raman spectroscopy (SERS) charged probes and an inverted superhydrophobic platform were used to develop a detection method for agricultural chemicals residues (ACRs) in rice combined with lightweight deep learning network. First, positively and negatively charged probes were prepared to adsorb ACRs molecules to SERS substrate. An inverted superhydrophobic platform was prepared to alleviate the coffee ring effect and induce tight self-assembly of nanoparticles for high sensitivity. Chlormequat chloride of 15.5-0.05 mg/L and acephate of 100.2-0.2 mg/L in rice were measured with the relative standard deviation of 4.15% and 6.25%. SqueezeNet were used to develop regression models for the analysis of chlormequat chloride and acephate. And the excellent performances were obtained with the coefficients of determination of prediction of 0.9836 and 0.9826 and root-mean-square errors of prediction of 0.49 and 4.08. Therefore, the proposed method can realize sensitive and accurate detection of ACRs in rice.

Asunto(s)

Aprendizaje Profundo , Nanopartículas del Metal , Oryza , Espectrometría Raman/métodos , Agroquímicos , Oryza/química , Clormequat , Nanopartículas del Metal/química , Interacciones Hidrofóbicas e Hidrofílicas

RESUMEN

As among the most widely used pesticides in agriculture, plant growth regulators (PGRs) have a positive influence on plants. However, the overuse of PGRs may induce toxicity in food and even be hazardous to human health. Numerous studies have investigated the presence and residues of PGRs on vegetables and fruits. Animal-derived foods are one of the most dominant food sources providing nutrients to fulfil the daily dietary intake, and could also be potentially contaminated by PGRs. However, there is little information on PGR residues in animal-derived foods. Standardization also lacks among the techniques for PGR determination in animal-derived foods, thereby restricting the further establishment of pesticide usage and food safety regulations. Therefore, in this study, a rapid and effective method for analyzing chlormequat chloride, thidiazuron, and paclobutrazol in animal-derived food samples was established. The method primarily involves high performance liquid chromatography-tandem mass spectrometry combined with the use of isotope-labeled internal standards. The extraction and clean-up procedures were based on the QuEChERS method. The analytes were extracted from pork, beef, chicken, pork liver, egg, and milk samples using acetonitrile, followed by 4 g anhydrous magnesium sulfate (MgSO4), and 1 g sodium chloride (NaCl). The supernatant was removed using a mixture of 50 mg N-propyl ethylenediamine (PSA), 50 mg octadecyl silane (C18), and 150 mg MgSO4, and then passed through a 0.22 µm membrane filter before determination. The Agilent ZORBAX Eclipse Plus C18 column (150 mm×3.0 mm, 1.8 µm) was used to separate the analytes under a gradient elution program, with acetonitrile and 5 mmol/L ammonium acetate solution as mobile phases. The analytes were detected by mass spectrometry using the positive and negative electrospray ionization modes under the multiple reaction monitoring mode. Matrix-matched calibration combined with internal standards was used to quantify the PGRs. The linear regression correlation coefficients (r2) for the PGRs were all greater than 0.990 in the corresponding linear concentration ranges. Chlormequat chloride, thidiazuron, and paclobutrazol showed good linearities in the range of 0.1-100 µg/L for the egg and pork liver samples and 0.1-50 µg/L for the pork, beef, and chicken samples. For the milk samples, thidiazuron and paclobutrazol showed good linearities in the range of 0.05-10 µg/L, while chlormequat chloride showed linearity in the range of 0.05-5 µg/L. The limit of detection (LOD) and limit of quantification (LOQ) for each PGR were based on the signal-to-noise (S/N) ratios. Under optimal conditions, the LODs ranged from 0.01 µg/kg to 0.1 µg/kg, where the LOD was defined as the amount of the tested compound that generated an S/N ratio higher than 3. In addition, the LOQs were in the range of 0.5-5 µg/kg, with an S/N ratio higher than 10. The precision and accuracy were evaluated by recovery experiments. At the LOQ, twice the LOQ, and 10 times the LOQ, the mean recoveries were in the range of 70.0%-117.4%, and the relative standard deviations (RSDs) ranged from 0.8% to 16.1%. The results indicated that the proposed method is accurate and reliable. This method is a modification of the QuEChERS method, and is advantageous owing to its simplicity and high sensitivity. The use of matrix-matching calibration curves and internal standards can eliminate matrix interference, thereby increasing the accuracy of the method. This method satisfies the testing requirements for chlormequat chloride, thidiazuron, and paclobutrazol residues in animal-derived foods, and is promising for the determination of other PGRs or other types of pesticides in animal-derived foods.

Asunto(s)

Reguladores del Crecimiento de las Plantas , Espectrometría de Masas en Tándem , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Humanos , Isótopos , Estándares de Referencia , Extracción en Fase Sólida

RESUMEN

Various generic extraction methods have been used to determine pesticide residues, mycotoxins, and polycyclic aromatic hydrocarbons (PAHs) in food and animal feed to ensure consumer safety. However, these methods cannot extract all relevant compounds at an acceptable rate of recovery. This study presents a new extraction method. This new method facilitated the identification of 231 compounds, including 196 pesticides, 11 mycotoxins, and 24 PAHs over a broad range of polarities. These compounds were identified in various sample matrices, including those that are lipid-rich. The processed sample is first extracted with water, acetonitrile, formic acid, and heptane. The addition of ammonium formate results in separation into three phases and enables analysis of the aqueous phase. Solid-phase extraction clean-up procedures were performed as necessary followed by analysis by liquid or gas chromatography and mass spectrometry. Analyte recoveries were typically in the range of 70 - 120% with relative standard deviations below 20%.

Asunto(s)

Micotoxinas/análisis , Residuos de Plaguicidas/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Alimentación Animal , Animales , Cromatografía Líquida de Alta Presión , Alimentos , Análisis de los Alimentos , Cromatografía de Gases y Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos

RESUMEN

Tebuconazole (TBZ) and Chlormequat chloride (CCC) combination has been established as highly effective in reducing plant height of lodging prone wheat varieties. In this work, a novel analytical method employing the quick, easy, cheap, effective, rugged and safe (QuEChERS) cleanup technique and LC-MS/MS (liquid chromatography-tandem mass spectroscopy) was developed for simultaneous estimation of TBZ and CCC in wheat grains and harvest stage plant leaves. A total of 10 mL of acetonitrile and 50 mg of primary secondary amine (PSA) sorbent was consumed in the optimized QuEChERS process for leaves and grain samples. The LC-MS/MS analysis was performed using a C-18 column operating under electrospray ionization in positive mode. The QuEChERS approach achieved extraction recoveries in the acceptable range of 70%-120%, for both the compounds and was validated in terms of accuracy, precision, sensitivity and linearity. Persistence study was conducted using Lihocin (CCC 50% SL), Folicur (TBZ 25.9% EC) and their combination tank mix (Lihocin + Folicur-50% SL + 25.9% EC) applied as foliar spray twice in wheat crop (tester tall variety C-306). The results demonstrated that the developed QuEChERS-LCMS/MS is rapid and confirmatory for simultaneous quantification of both the test analytes in wheat crop.

Asunto(s)

Fraccionamiento Químico/métodos , Clormequat/análisis , Espectrometría de Masas en Tándem/métodos , Triazoles/análisis , Triticum/química , Acetonitrilos , Agricultura/métodos , Cromatografía Liquida/métodos , Productos Agrícolas/química , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Hojas de la Planta/química , Reproducibilidad de los Resultados , Semillas/química , Sensibilidad y Especificidad

RESUMEN

In this study, we analyze changing patterns of pesticide use in agriculture in Great Britain over the 1990-2016 period, with respect to the risk they pose to birds. The weight of pesticides applied decreased by 51% between 1990 and 2016, but the area treated increased by 63% over the same period. Over this period, there has been considerable turnover in the pesticides used. The European Union (including Great Britain until 2020) has restricted or banned many pesticides for agricultural use, including organophosphates and carbamates. However, new generations of active substances have been introduced, such as the neonicotinoids, some of which have since been banned. In this analysis, we estimate the annual 'toxic load' of agricultural pesticide use in Great Britain for birds, measured as the total number of LD50 doses for corn buntings, Emberiza calandra. We have previously performed similar analyses for bees, for which the total toxic load increased six-fold during this period. In contrast, for birds the total toxic load fell by 80.5%, although still correspond to 8.3e+11 corn bunting LD50 doses in 2016. The decrease in toxicity is largely due to declining use of highly toxic organophosphates in recent years. We identify the pesticides in current use that may pose the highest risk to birds, which include a mix of insecticides, herbicides, fungicides, molluscicides, acaricides and plant growth regulators. The insecticide ethoprop was ranked highest in 2016, with a toxic load of 71 billion potential corn bunting kills. Some of the other chemicals presenting a high toxic load, such as the herbicide chlormequat, are not highly toxic to birds (in terms of LD50) but are used in very large quantities. However, it is important to stress that, in reality, only a tiny proportion of pesticides applied will be ingested by birds, and this will vary according to timing and method of application, persistence of the active substance and many other factors. We further note that impacts of pesticides on birds might often be indirect, for example via depleting their food supply, and that sublethal impacts may occur at much lower doses than the LD50, neither of which do we investigate here. Nonetheless, we suggest that this is a useful approach to highlight pesticides that might be worth closer study with regard to possible impacts.

RESUMEN

We showed previously that chlormequat chloride, a widely used plant growth regulator, could affect embryonic growth and growth hormone (GH)-insulin-like growth factor 1 (IGF-1) axis of rats. However, the potential effects of low dose chlormequat chloride exposure during pregnancy on embryonic and postnatal growth and development remain unclear. To further assess the risk of chlormequat chloride to human embryonic growth and postnatal health, we exposed maternal rats orally to the chemical during pregnancy at 5 mg/kg bw, a dose corresponding to the human acceptable daily intake (ADI) level set by World Health Organization (WHO), and determined the effects of chlormequat on embryo growth and postnatal health. We found that chlormequat chloride increased embryonic growth parameters, GH, and GH-releasing hormone (GHRH) levels, but did not affect somatostatin and IGF-1 on gestational day (GD) 11. In the pups of postnatal day (PD) 7, we observed increased head length, decreased body fat percentage, hypoglycemia, hyperlipidemia and hyperproteinemia. In conclusion, maternal exposure to chlormequat chloride during pregnancy disrupts the embryonic growth probably through its effects on growth regulators and even has adverse effects on postnatal health.

Asunto(s)

Anomalías Inducidas por Medicamentos/patología , Clormequat/toxicidad , Desarrollo Embrionario/efectos de los fármacos , Animales , Animales Recién Nacidos , Composición Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/biosíntesis , Hormona Liberadora de Hormona del Crecimiento/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Masculino , Exposición Materna , Reguladores del Crecimiento de las Plantas/toxicidad , Embarazo , Ratas , Ratas Sprague-Dawley , Somatostatina/biosíntesis

RESUMEN

The study reports the role of choline and compounds thereof in the formation of chlormequat under thermal conditions, with emphasis on the molecular mechanism involved in the transformation. The data show the decomposition of choline to chlormequat at 200 °C in presence of chloride ions, likely by nucleophilic substitution. Furthermore, the results suggest that phosphatidylcholine, glycerophosphocholine, and phosphocholine are the effective precursors of chlormequat under sufficient thermal conditions due to their capability to degrade to choline and/or the ability of the phosphate moiety to behave as a good leaving group with respect to nucleophilic attacks. Thermal treatments (120 and 200 °C) applied to egg powder, rich in phosphatidylcholine, and wheat flour, with choline at a substantial level, suggest that less energy is required for obtaining chlormequat from phosphatidylcholine than from choline. This observation is consistent with the postulated mechanism of a nucleophilic substitution with phosphate moieties acting as better leaving groups than the hydroxyl group.

Asunto(s)

Clormequat/análisis , Huevos/análisis , Harina/análisis , Reguladores del Crecimiento de las Plantas/análisis , Triticum/química , Animales , Pollos , Colina/análisis , Calor

RESUMEN

Chlormequat chloride, a plant growth regulator, is widely applied in agriculture because it can promote sturdier growth of the crops. In this research, we found that rat embryo growth on GD11 was inhibited in vitro at 50 µg/ml but promoted in vivo at 75 mg/kg.bw by maternal oral exposure. Therefore, the concentrations of chlormequat chloride in the sera of the pregnant rats on gestation day (GD)11 were determined by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) test to be 1.94 ± 0.023 µg/ml, 3.84 ± 0.080 µg/ml, and 7.08 ± 0.11 µg/ml, respectively, when the pregnant rats were orally exposed to chlormequat chloride at 75, 137.5, and 200 mg/kg.bw. Hence, we performed WEC tests again and confirmed that the rat embryo growth in vitro was promoted by chlormequat chloride at 5 µg/mL. The embryonic growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels were increased by chlormequat chloride both in vitro and in vivo compared with the control ones. We concluded that chlormequat chloride could elevate GH and IGF-1 levels in embryos and promote embryonic growth both in vitro and in vivo.

Asunto(s)

Clormequat/farmacología , Desarrollo Embrionario/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Animales , Clormequat/administración & dosificación , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Femenino , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Embarazo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem

RESUMEN

A method for the determination of chlormequat chloride (CCC) residues in animal derived foods by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The samples were extracted with acetonitrile containing 1% (v/v) acetic acid and defatted with n-hexane, followed by clean-up on a cationic solid phase extraction column. The analytes were separated on a Venusil MP C18(2) column (150 mm×2.1 mm, 3 µm) under a gradient elution program using acetonitrile and 0.1% (v/v) formic acid aqueous solution as the mobile phases. Then, the analytes were detected by tandem mass spectrometry using a positive electrospray ionization (ESI+) source in the multiple reaction monitoring (MRM) mode. Matrix-matched internal standard calibration curves were used for quantitative analysis. The calibration curves showed good linearity in the range of 0.200-500 µg/L for CCC, with correlation coefficients (r2) no less than 0.9993. The limit of quantification (LOQ) of the method was 0.500 µg/kg. The average recoveries of CCC in pork, beef, mutton, chicken, egg, pig kidney, beef liver, sheep kidney, chicken liver and milk matrices at spiked levels of 0.500-500 µg/kg were 93.4%-101%, and the relative standard deviations were 2.3%-8.0%. The method has less matrix interference, with high sensitivity, accuracy and reliability, and it is suitable for the quantitative detection of CCC residues in animal derived foods.

Asunto(s)

Clormequat/análisis , Huevos/análisis , Análisis de los Alimentos , Carne Roja/análisis , Animales , Cromatografía Líquida de Alta Presión , Leche , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Espectrometría de Masas en Tándem

RESUMEN

The European Commission requested EFSA to provide a statement in the framework of Article 43 of Regulation (EC) No 396/2005 on the dietary risk assessment for the proposed temporary maximum residue levels (MRLs) (6 and 7 mg/kg) for chlormequat in cultivated oyster mushrooms. The MRL proposals were derived by the evaluating Member State (EMS) Germany. Chlormequat residues can be found in mushrooms due to cross-contamination from cereal straw lawfully treated with chlormequat chloride which is used as cultivation substrate. EFSA concluded that the exposure to residue levels at the proposed MRLs is unlikely to pose a risk to consumers' health. However, EFSA recommended to take appropriate risk management actions to avoid contamination of cultivated oyster mushrooms and other fungi cultivated on straw.

RESUMEN

This study described the development and validation of a simple, rapid, specific and sensitive method for detecting chlormequat chloride (CQ) and mepiquat chloride (MQ) residues in tomato cultivation matrices covering soil, water, seedling samples. The dissipation rates of CQ and MQ in tomato cultivation matrices were also determined in this study. A Hydrophilic Interaction Liquid Chromatography (HILIC) column was used for chromatographic separation. A triple quadrupole mass spectrometer equipped with an electrospray ionisation source in positive ion mode by multiple reaction monitoring was used for detection. Soil samples were extracted with accelerated solvent extraction (ASE) and cleaned up with WCX phase extraction column; water samples were extracted with WCX phase extraction column; seedling samples were extracted with methanol-ammonium acetate solution. LODs and LOQs of CQ and MQ were 0.02µg/kg and 0.1µg/kg in soil samples, 0.005ng/mL and 0.02ng/mL in water samples, and 0.05µg/kg and 1.0µg/kg in seedling samples, respectively. The mean recovery rate of CQ in soil, water and seedling samples ranged from 76.98% to 111.60%. While the mean recovery rate of MQ in soil, water and seedling samples ranged from 96.90% to 105.40%. The fastest to the slowest metabolising rates of CQ and MQ were as follows: soil samples>seedling samples>water samples. In conclusion, this study provided a new potential method for detecting CQ and MQ in tomato cultivation matrices using ultra-performance liquid chromatography-tandem mass spectrometry.

Asunto(s)

Clormequat/análisis , Cromatografía Líquida de Alta Presión/métodos , Residuos de Medicamentos/análisis , Piperidinas/análisis , Suelo/química , Solanum lycopersicum/química , Espectrometría de Masas en Tándem/métodos , Agricultura , Clormequat/química , Clormequat/aislamiento & purificación , Límite de Detección , Modelos Lineales , Piperidinas/química , Piperidinas/aislamiento & purificación , Reguladores del Crecimiento de las Plantas/análisis , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/aislamiento & purificación , Reproducibilidad de los Resultados , Plantones/química , Extracción en Fase Sólida/métodos

RESUMEN

Chlormequat Chloride (CCC), a widely used plant growth regulator, could decrease body weight in animals; however, the mechanism has not been well studied. This study was designed to evaluate the skeletal development toxicity of CCC on pubertal male Sprague-Dawley (SD) rats and to investigate whether CCC impacts the development of chondrocyte, osteoblast and osteoclast through growth hormone (GH) and insulin like growth factor 1 (IGF-I). Rats from 23 to 70 on postnatal days were exposed to CCC daily by gavage at doses of 0, 75, 150, and 300mg/kg bw/d. The results showed that the size of femurs and tibias, bone mineral density and biomechanical parameters were significantly decreased in the 300mg/kg bw/d group compared with the control group. The concentration of osteocalcin (OCN) and C-terminal telopeptide of type I collagen (CTX-I) in blood in the 150mg/kg bw/d group was also changed. The mRNA expression ratio of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in 150 and 300mg/kg bw/d group was increased. Histological analysis of proximal and distal epiphyseal plates of the right femurs showed that both the proliferative zone and hypertrophic zone narrowed in CCC-treated groups. The concentration of IGF-I in blood was reduced with an increase in exposure doses of CCC. The mRNA expression of growth hormone receptor (GHR) in tibia was decreased in the CCC-treated group. The results indicated that CCC might indirectly impact the formation and activation of chondrocytes, osteoblasts and osteoclasts because of the decline of GHR and IGF-I, leading to skeletal development damage.

Asunto(s)

Clormequat/toxicidad , Condrocitos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Esqueleto/efectos de los fármacos , Animales , Peso Corporal , Densidad Ósea/efectos de los fármacos , Condrocitos/metabolismo , Colágeno Tipo I/sangre , Relación Dosis-Respuesta a Droga , Fémur/efectos de los fármacos , Fémur/metabolismo , Hormona del Crecimiento/metabolismo , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Osteocalcina/sangre , Osteoclastos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Péptidos/sangre , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Esqueleto/crecimiento & desarrollo , Tibia/efectos de los fármacos , Tibia/metabolismo

RESUMEN

Chlormequat Chloride (CCC) is a plant growth regulator that is widely applied in agriculture. Previous studies have shown that long-term exposure of CCC could decrease body weight in animals. However, the underlying mechanisms have not been studied. In this study, CCC was administered to rats daily by gavage on postnatal days 23-60 at doses of 0, 75, 150 and 300mg/kg bw/d. The results showed that body weight and the length of the right femur were significantly decreased in the 300mg/kg bw/d group. Histological analysis of proximal growth plates of the right femurs showed narrowed proliferative zones and hypertrophic zones in CCC-treated groups. The mRNA expression of growth hormone, growth hormone receptor and insulin like growth factor 1 were decreased in the CCC-treated group. The results indicated that CCC may affect the expression of growth hormone and insulin-like growth factor 1 and subsequently cause a decrease in body weight and bone length.

Asunto(s)

Peso Corporal/efectos de los fármacos , Clormequat/toxicidad , Hormona del Crecimiento/sangre , Acetilcolina/sangre , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/genética , Placa de Crecimiento/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Tamaño de los Órganos/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/toxicidad , Pubertad/efectos de los fármacos , Ratas Sprague-Dawley , Receptores de Somatotropina/genética