Dezhen Wang

Evaluating the enantioselective degradation and novel metabolites following a single oral dose of metalaxyl in mice.

Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-D,L-alaninemethylester] is a systemic fungicide widely used in agriculture. In this study, the enantioselective distribution, degradation and excretion of metalaxyl were investigated after oral gavage administration of rac-metalaxyl to mice. Concentration of metalaxyl and its enantiomers was determined by HPLC-MS/MS. The results showed that R-metalaxyl was much higher than S-metalaxyl in heart, liver, lung, urine and feces. As for the strong first pass effect, concentrations of metalaxyl in liver were much higher than those in other tissues. The total body clearance (CL) of metalaxyl in mice was 1.77 L h(-1 )kg(-1) and degradation half-lives of (t1/2) of S-metalaxyl and R-metalaxyl in liver were 2.2 h and 3.0 h, respectively. Such results indicated the enantioselectivity of metalaxyl lies in distribution, degradation and excretion processes in mice. Main metabolites were also determined and biotransformation reactions were hydroxylation, demethylation and didemethylation. Furthermore, metabolite concentrations in urine and feces were much higher than those in tissues. These results may have potential implications to predict toxicity and provide additional information associated with adverse health effects for risk assessment of metalaxyl.

NMR- and LC–MS/MS-based urine metabolomic investigation of the subacute effects of hexabromocyclododecane in mice

In the present study, both untargeted and targeted metabolomics approaches were used to evaluate the subacute effects of hexabromocyclododecane (HBCD) on mice urine metabolome. Untargeted metabolomics based on 1H NMR showed that HBCD exposure disturbed mice metabolism in both dosed groups, especially in high dosed group. The low-dose HBCD led to a decrease in alanine, malonic acid, and trimethylamine (TMA). High-dose HBCD-treated mice developed high levels of citric acid and 2-ketoglutarate, together with decreased alanine, acetate, formate, TMA, 3-hydroxybutyrate, and malonic acid. Targeted metabolomics for metabolic profiling of 20 amino acids identified alanine, lysine, and phenylalanine as significantly disturbed metabolites. These results indicated that subchronic exposure to HBCD caused a disturbance of mice metabolism, especially in TCA cycle, lipid metabolism, gut microbial metabolism, and homeostasis of amino acids, and the application of untargeted and targeted metabolomics combined with conventional toxicology approaches to evaluate the subacute effects of pollutants will provide more comprehensive information and aid in predicting health risk of these pollutants.

Enantioselective Metabolism and Interference on Tryptophan Metabolism of Myclobutanil in Rat Hepatocytes

Myclobutanil, (RS)-2-(4-chlorophenyl)-2-(1H-1, 2, 4-triazol-1-ylmethyl) hexanenitrile is a widely used triazole fungicide. In this study, enantioselective metabolism and cytotoxicity were investigated in rat hepatocytes by chiral HPLC-MS/MS and the methyl tetrazolium (MTT) assay, respectively. Furthermore, tryptophan metabolism disturbance in rat hepatocytes after myclobutanil exposure was also evaluated by target metabolomics method. The half-life (t1/2) of (+)-myclobutanil was 10.66 h, whereas that for (-)-myclobutanil was 15.07 h. Such results indicated that the metabolic process of myclobutanil in rat hepatocytes was enantioselective with an enrichment of (-)-myclobutanil. For the cytotoxicity research, the calculated EC50 (12 h) values for rac-myclobutanil, (+)- and (-)-myclobutanil were 123.65, 150.65 and 152.60 µM, respectively. The results of tryptophan metabolites profiling showed that the levels of kynurenine (KYN) and XA were both up-regulated compared to the control, suggesting the activation effect of the KYN pathway by myclobutanil and its enantiomers which may provide an important insight into its toxicity mechanism. The data presented here could be useful for the environmental hazard assessment of myclobutanil.

Enantiomeric Separation of Chiral Pesticides by Permethylated β-Cyclodextrin Stationary Phase in Reversed Phase Liquid Chromatography.

Enantiomeric separation of six chiral pesticides by high-performance liquid chromatography with permethylated β-cyclodextrin (β-PM) chiral stationary phase were tested under reversed phase conditions. The influences of water composition from 10% to 45% in the mobile phase and column temperatures from 0°C to 40°C on the separation were investigated. Baseline separation was obtained for diclofop-methyl, fenoxaprop-ethyl, tebuconazole and triticonazole, and Rs of these pesticides were greater than 1.5. However, etoxazole and lactofen were partially separated in all experiments.

Effects of the bioconcentration and parental transfer of environmentally relevant concentrations of difenoconazole on endocrine disruption in zebrafish (Danio rerio)

Difenoconazole, a typical triazole fungicide, inhibits lanosterol-14R-demethylase (CYP51) to prevent fungal sterol synthesis and its residues are frequently detected in the environment due to its wide application. Previous studies have demonstrated that difenoconazole altered the triglyceride levels, and gene expression relevant to cholesterol biosynthesis in zebrafish. However, endocrine-disruption in the hypothalamus-pituitary-gonadal-liver (HPGL) axis, the effects of transferring to offspring, and the underlying mechanisms of difenoconazole in aquatic organisms are still unknown. In this study, we defined the effects of difenoconazole at environmental concentrations on endocrine disturbance using zebrafish as an experimental model. The results indicated that difenoconazole induced a significant change in the somatic index, and pathological variations in tissues, and steroid hormone levels. RT-PCR experiments further confirmed that difenoconazole significantly induced expression alteration of lhr, hsd3β, hsd11β, cyp19a in the ovary and star, cyp19a, cyp3c1 in the testis, and erα genes in livers. In addition, difenoconazole exposure in parental zebrafish affected the hatchability and length of its offspring. Moreover, the burdens of difenoconazole and difenoconazole alcohol in females were higher than in males. These findings highlighted that difenoconazole exposure at environmentally relevant concentrations elicited estrogenic endocrine-disruption effects via altering homeostasis of sex steroid hormones in the HPGL axis and the adverse effects can be transferred to the offspring.

Enantioselective Effects of Metalaxyl Enantiomers on Breast Cancer Cells Metabolic Profiling Using HPLC-QTOF-Based Metabolomics

In this study, an integrative high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF) based metabolomics approach was performed to evaluate the enantioselective metabolic perturbations in MCF-7 cells after treatment with R-metalaxyl and S-metalaxyl, respectively. Untargeted metabolomics profile, multivariate pattern recognition, metabolites identification, and pathway analysis were determined after metalaxyl enantiomer exposure. Principal component analysis (PCA) and partitial least-squares discriminant analysis (PLS-DA) directly reflected the enantioselective metabolic perturbations induced by metalaxyl enantiomers. On the basis of multivariate statistical results, a total of 49 metabolites including carbohydrates, amino acids, nucleotides, fatty acids, organic acids, phospholipids, indoles, derivatives, etc. were found to be the most significantly changed metabolites and metabolic fluctuations caused by the same concentration of R-metalaxyl and S-metalaxyl were enantioselective. Pathway analysis indicated that R-metalaxyl and S-metalaxyl mainly affected the 7 and 10 pathways in MCF-7 cells, respectively, implying the perturbed pathways induced by metalaxyl enantiomers were also enantioselective. Furthermore, the significantly perturbed metabolic pathways were highly related to energy metabolism, amino acid metabolism, lipid metabolism, and antioxidant defense. Such results provide more specific insights into the enantioselective metabolic effects of chiral pesticides in breast cancer progression, reveal the underlying mechanisms, and provide available data for the health risk assessments of chiral environmental pollutants at the molecular level.

Sex-specific effects of difenoconazole on the growth hormone endocrine axis in adult zebrafish (Danio rerio)

Difenoconazole, as one of the most widely used triazole fungicides, is applied to protect crops, fruits, and vegetables. It has been reported that difenoconazole can enter the environment and impair aquatic organisms, but whether difenoconazole can disrupt the growth hormone (GH) balance in adult zebrafish (Danio rerio) is still unclear. In this study, adult female and male zebrafish were exposed to difenoconazole (0, 5, 50, and 500µg/L) for seven days. The results revealed that the bioaccumulation of difenoconazole and its primary metabolite difenoconazole alcohol in females were both larger than that in males. In females, the growth of the liver and ovary were inhibited, which may be due to the decreased transcription of the key genes igf1a, igf2a, and igf2b in both organs. Male fish growth was promoted in response to the increased expression of genes relevant to the GH/insulin-like growth factor axis (GH/IGF) axis in the brain, liver, and testis as well as increased GH levels. It was found that difenoconazole interfered with the growth endocrine system and sex-specifically altered the expression of GH/IGF axis related genes in adult zebrafish after a short-term exposure.

Subacute oral toxicity assessment of benalaxyl in mice based on metabolomics methods

In this study, the metabolic responses of mice after 30 days of exposure to benalaxyl were assessed using NMR-based untargeted metabolomics and LC-MS-based targeted profiling of 20 amino acids. Urinary 1H NMR analyses revealed alterations in energy metabolism, lipid metabolism, vitamin B metabolism, the urea cycle and amino acid metabolism, and targeted analyses indicated that the serum levels of asparagine, histidine, lysine and aspartic acid were significantly altered. Additionally, significant oxidative stress was observed in the liver and kidney, although no apparent histopathological injury was observed. The tissue distribution indicated a significant stereoselectivity in the brain, where (-)-R-benalaxyl was enriched. These data provide a comprehensive picture of the subacute toxic effects of benalaxyl in mice. The results of this study suggested that, for a toxicity evaluation, metabolomics analysis is much more sensitive than traditional toxicological methods. The results also highlight the combined use of untargeted and targeted metabolomics approaches in evaluating the health risks of xenobiotics.

Metabolomics and transcriptomics reveal the toxicity of difenoconazole to the early life stages of zebrafish ( Danio rerio )

Difenoconazole is widely used to inhibit the growth of fungi, but its residue in the water environment may threaten ecosystem and human health. Here, 1H nuclear magnetic resonance (NMR) and LC-MS/MS based metabolomics and transcriptomics approaches were used to assess the response of zebrafish to difenoconazole exposure. Early life stages of zebrafish were exposed to difenoconazole at environmentally relevant concentrations for 168h. Their comparison with the control group suggested an adverse development and disturbance of steroid hormones and VTG. KEGG pathway analysis identified five biological processes on the basis of differentially expressed genes (DEGs), as well as altered metabolites and amino acids in zebrafish following difenoconazole exposure. These affected processes included energy metabolism, amino acids metabolism, lipid metabolism, nucleotide metabolism, and an immune-related pathway. Collectively, these results bring us closer to an incremental understanding of the toxic effects of difenoconazole on zebrafish in its early development, and lend support to the continued use of the early life stages of zebrafish as a classical model to evaluate underlying environmental risks of xenobiotics in aquatic organisms.

Evaluating the enantioselective distribution, degradation and excretion of epoxiconazole in mice following a single oral gavage

Abstract 1. The enantiomeric enrichment or degradation of epoxiconazole has been reported in grape, soil, tubifex and mealworm beetle. But, little is known about its enantioselective behaviors in mammals. 2. To further understand differences in the distribution, degradation and excretion of epoxiconazole enantiomers in vivo, male CD-1 mice were selected as the test model to investigate the enantioselective behaviors after a single oral gavage. Mice were sacrificed after 1 h, 3 h, 7 h, 12 h, 24 h, 48 h, 72 h treatment, blood, tissues and excretions were collected for epoxiconazole analysis by LC-MS/MS. 3. On the Lux-Cellulose-1 chiral column, an enrichment of the second eluting (+)-epoxiconazole was generally observed, and feces and urine showed similar EF with major tissues. 4. To elucidate the potential role of intestinal bacterial flora in stereospecific degradation of epoxiconazole, mice fecal flora were cultured in vitro and incubated with epoxiconazole for 48 h. Results showed that (−)-epoxiconazole was preferentially degraded by intestinal bacterial. 5. These results may provide useful information for risk assessment of epoxiconazole on non-target animals.