Chlorocholine chloride exposure induced spermatogenic dysfunction via iron overload caused by AhR/PERK axis-dependent ferritinophagy activation.

Chlorocholine chloride (CCC) is a plant growth regulator used worldwide that is detectable in cereals, fruits and animal products. The health effects of CCC exposure have raised public concern. Our previous research showed that CCC exposure decreased testosterone synthesis in pubertal rats. However, little is known about whether and how pubertal CCC exposure impacts spermatogenesis. In this study, we used BALB/c mice and spermatogonia-derived GC-1 cells to examine CCC-induced spermatogenic dysfunction. In vivo, pubertal CCC exposure led to decreased testicular weight, decreased testicular germ cells and poor sperm quality. This effect worsened after cessation of CCC exposure for the next 30 days. RNA-seq and western blot analysis revealed that CCC induced aryl hydrocarbon receptor (AhR) signaling, endoplasmic reticulum stress (ERS) and ferritinophagy. Increased iron content and lipid peroxidation levels were also observed in CCC-treated testes. In vitro, it was identified that iron overload mediated by enhanced ferritinophagy occurred in CCC-treated GC-1 cells, which might be attributed to the PERK pathway in ERS. Further, for the first time, our study elucidated the involvement of AhR in CCC-induced iron overload, which aggravated testicular oxidative damage via lipid peroxidation. Considering the adverse impact of CCC exposure on rodents, supportive evidence from GC-1 cells, and the critical importance of spermatogenesis on male development, the effects of CCC on the male reproduction warrant increased attention.

Chlormequat chloride induced activation of calmodulin mediated PI3K/AKT signaling pathway led to impaired sperm quality in pubertal mice.

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.

Mixed Compound of DCPTA and CCC Increases Maize Yield by Improving Plant Morphology and Up-Regulating Photosynthetic Capacity and Antioxidants.

DCPTA (2-diethylaminoethyl-3, 4-dichlorophenylether) and CCC (2-chloroethyltrimethyl- ammonium chloride) have a great effect on maize growth, but applying DCPTA individually can promote the increase of plant height, resulting in the rise of lodging percent. Plant height and lodging percent decrease in CCC-treated plants, but the accumulation of biomass reduce, resulting in yield decrease. Based on the former experiments, the performance of a mixture which contained 40 mg DCPTA and 20 mg CCC as active ingredients per liter of solution, called PCH was tested with applying 40mg/L DCPTA and 20mg/L CCC individually. Grain yield, yield components, internode characters, leaf area per plant, plant height and lodging percent as well as chlorophyll content, chlorophyll fluorescence, enzymatic antioxidants, membranous peroxide and organic osmolyte were analyzed in two years (2011 and 2012), using maize hybrid, Zhengdan 958 (ZD 958) at density of 6.75 plants m-2. CCC, DCPTA and PCH were sprayed on the whole plant leaves at 7 expanded leaves stage and water was used as control. Compared to control, PCH significantly increased grain yield (by 9.53% and 6.68%) from 2011 to 2012. CCC significantly decreased kernel number per ear (by 6.78% and 5.69%) and thousand kernel weight (TKW) (by 8.57% and 6.55%) from 2011 to 2012. Kernel number per ear and TKW increased in DCPTA-treated and PCH-treated plants, but showed no significant difference between them. In CCC-treated and PCH-treated plants, internode length and plant height decreased, internode diameter increased, resulting in the significant decline of lodging percent. With DCPTA application, internode diameter increased, but internode length and plant height increased at the same time, resulting in the augment of lodging percent. Bending strength and puncture strength were increased by applying different plant growth regulators (PGRs). In PCH-treated plants, bending strength and puncture strength were greater than other treatments. Compared to control, the bending strength of 3rd internode was increased by 14.47% in PCH-treated plants in 2011, increased by 18.40% in 2012, and the difference was significant. Puncture strength of 1st, 3rd and 5th internode was increased by 37.25%, 29.17% and 26.09% in 2011 and 34.04%, 25% and 23.68% in 2012, compared to control. Leaf area and dry weight per plant reduced significantly in CCC-treated plants, increased in DCPTA-treated and PCH-treated plants from 2011 to 2012. Chlorophyll content and chlorophyll fluorescence improved with CCC and DCPTA application. Due to the additive effect of DCPTA and CCC, PCH showed the significant effect on chlorophyll content and chlorophyll fluorescence. Compared to control, total enzyme activity (SOD, POD, CAT, APX and GR) and soluble protein content increased, malonaldehyde (MDA) and hydrogen peroxide (H2O2) content reduced in PCH-treated plants. The transportation of soluble sugar from leaf to kernel improved significantly at the late silking stage. The research provided the way for the further use of DCPTA and CCC into the production practice.

Fate and transport of chlormequat in subsurface environments.

BACKGROUND, AIM AND SCOPE: Chlormequat (Cq) is a plant growth regulator used throughout the world. Despite indications of possible effects of Cq on mammalian health and fertility, little is known about its fate and transport in subsurface environments. The aim of this study was to determine the fate of Cq in three Danish subsurface environments, in particular with respect to retardation of Cq in the A and B horizons and the risk of leaching to the aquatic environment. The study combines laboratory fate studies of Cq sorption and dissipation with field scale monitoring of the concentration of Cq in the subsurface environment, including artificial drains. MATERIALS AND METHODS: For the laboratory studies, soil was sampled from the A and B horizons at three Danish field research stations-two clayey till sites and one coarse sandy site. Adsorption and desorption were described by means of the distribution coefficient (K (d)) and the Freundlich adsorption coefficient (K (F,ads)). The dissipation rate was estimated using soil sampled from the A horizon at the three sites. Half life (DT(50)) was calculated by approximation to first-order kinetics. A total of 282 water samples were collected at the sites under the field monitoring study- groundwater from shallow monitoring screens located 1.5-4.5 m b.g.s. at all three sites as well as drainage water from the two clayey sites and porewater from suction cups at the sandy site, in both cases from 1 m b.g.s. The samples were analysed using LC-MS/MS. The field monitoring study was supported by hydrological modelling, which provided an overall water balance and a description of soil water dynamics in the vadose zone. RESULTS: The DT(50) of Cq from the A horizon ranged from 21 to 61 days. The Cq concentration-dependant distribution coefficient (K (d)) ranged from 2 to 566 cm(3)/g (median 18 cm(3)/g), and was lowest in the sandy soil (both the A and B horizons). The K (F,ads) ranged from 3 to 23 (microg(1 - 1/n ) (cm(3))(1/n) g(-1)) with the exponent (1/n) ranging from 0.44 to 0.87, and was lowest in the soil from the sandy site. Desorption of Cq was very low for the soil types investigated (<10%w). Cq in concentrations exceeding the detection limit (0.01 microg/L) was only found in two of the 282 water samples, the highest concentration being 0.017 microg/L. DISCUSSION: That sorption was highest in the clayey till soils is attributable to the composition of the soil, the soil clay and iron content being the main determinant of Cq sorption in both the A and B horizons of the subsurface environment. Cq was not detected in concentrations exceeding the detection limit in either the groundwater or the porewater at the sandy site. The only two samples in which Cq was detected were drainage water samples from the two clayey till sites. The presence of Cq here was probably attributable to the hydrogeological setting as water flow at the two clayey till sites is dominated by macropore flow and less by the flow in the low permeability matrix. In contrast, water flow at the sandy site is dominated by matrix flow in the high permeability matrix, with negligible macropore flow. Given the characteristics of these field sites, Cq adsorption and desorption can be expected to be controlled by the clay composition and content and the iron content. Combining these observations with the findings of the sorption and dissipation studies indicates that the key determinant of Cq retardation and fate in the soil is sorption characteristics and bioavailability. CONCLUSIONS: The leaching risk of Cq was negligible at the clayey till and sandy sites investigated. The adsorption and desorption experiments indicated that absorption of Cq was high at all three sites, in particular at the clayey till sites, and that desorption was generally very limited. The study indicates that leaching of Cq to the groundwater is hindered by sorption and dissipation. The detection of Cq in drainage water at the clayey till sites and the evidence for rapid transport through macropores indicate that heavy precipitation events may cause pulses of Cq. RECOMMENDATIONS AND PERSPECTIVES: The present study is the first to indicate that the risk of Cq leaching to the groundwater and surface water is low. Prior to any generalisation of the present results, the fate of Cq needs to be studied in other soil types, application regimes and climatic conditions to determine the Cq retardation capacity of the soils. The study identifies bioavailability and heavy precipitation events as important factors when assessing the risk of Cq contamination of the aquatic environment. The possible effects of future climate change need to be considered when assessing whether or not Cq poses an environmental risk.

Sudden death following accidental ingestion of chlormequat.

A 59-year-old white male accidentally ingested a mouthful of a plant growth chemical, Cycocel, containing 11.8% of the active ingredient (2-chloroethyl)trimethylammonium chloride (chlormequat). He was seen by a family physician and then transferred to a hospital where he died as a result of ventricular fibrillation, which progressed to asystole. Postingestion symptoms were typical of cholinergic crisis and included salivation, diaphoresis, bradycardia, visual disturbances, and seizure. Autopsy findings showed marked pulmonary edema, coronary atherosclerosis, atheromata of aorta, and localized adenocarcinoma of the prostate. Toxicological analyses of biological samples showed the presence of chlormequat in the stomach contents and urine.

[In-vitro studies on the break-down of chlormequat (chlorcholinchloride) in the rumen]. / In-vitro-Untersuchungen zum Abbau von Chlormequat (Chlorcholinchlorid) im Pansen

Under anaerobic conditions chlormequat did not undergo microbial breakdown in samples of rumen contents or rumen juice. Comparison with other forms of microbial breakdown of chlormequat, such as in soil, showed that oxidative processes were responsible.

[Occurrence of chlormequat (CCC) in wheat straw and its excretion in milk following feeding of straw pellets containing chlormequat]. / UPNTERSUCHUNGEN ZUM Vorkommen von Chlormequat (CCC) in Weizenstroh und seine Exkretion mit der Milch nach Verfütterung chlormequathaltiger strohpellets

When the herbicide chlormequat (chlorocholine chloride or CCC) was correctly used at 2 kg per hectare at the five-leaf stage of growth, the residues in wheat straw did not exceed 3 ppm. Such residual amounts remained unchanged after storage and pelleting. However, the feeding of contaminated straw pellets to cows did not result in contaimination of the milk detectable by a method which was capable of determining concentrations down to 0.05 ppm.