ABSTRACT:Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a broad-spectrum chlorinated cyclodiene insecticide. This study was performed to elucidate the stereoselective metabolism of endosulfan in human liver microsomes and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of endosulfan. Human liver microsomal incubation of endosulfan in the presence of NADPH resulted in the formation of the toxic metabolite, endosulfan sulfate. The intrinsic clearances (CL int ) of endosulfan sulfate from ␤-endosulfan were 3.5-fold higher than those from ␣-endosulfan, suggesting that ␤-endosulfan would be cleared more rapidly than ␣-endosulfan. Correlation analysis between the known P450 enzyme activities and the rate of the formation of endosulfan sulfate in the 14 human liver microsomes showed that ␣-endosulfan metabolism is significantly correlated with CYP2B6-mediated bupropion hydroxylation and CYP3A-mediated midazolam hydroxylation, and that ␤-endosulfan metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in human liver microsomes and the incubation study of cDNA-expressed enzymes also demonstrated that the stereoselective endosulfan sulfate formation from ␣-endosulfan is mediated by CYP2B6, CYP3A4, and CYP3A5, and that from ␤-endosulfan is mediated by CYP3A4 and CYP3A5. The total CL int values of endosulfan sulfate formation catalyzed by CYP3A4 and CYP3A5 were consistently higher for ␤-endosulfan than for the ␣-form (CL int of 0.67 versus 10.46 l/min/pmol P450, respectively). CYP2B6 stereoselectively metabolizes ␣-endosulfan, but not ␤-endosulfan. These findings suggest that the CYP2B6 and CYP3A enzymes are major enzymes contributing to the stereoselective disposition of endosulfan.

Lindane is a broad-spectrum persistent organochlorine pesticide that has been used to control pests for many years. In this study, its toxic mechanisms in adult zebrafish were investigated using targeted metabolomics with GC-MS/MS and non-targeted metabolomics with LC-Orbitrap-MS/MS. Zebrafish was exposed to lindane in water for 48 h in three groups: control, low exposure (1/10 LC50) and high exposure (LC50). In the zebrafish exposed to low concentration of lindane, 2.24–3.98 mg/kg of lindane were determined, while 35.67–56.46 mg/kg were observed in the zebrafish exposed to high concentration. A total of 118 metabolites were identified from 394 metabolites on GC-MS/MS and 45 metabolites were selected as biomarkers. A total of 62 metabolites were identified on LC-Orbitrap-MS/MS and 7 metabolites were selected as biomarkers. Three groups were well separated on partial least squares-discriminant analysis (PLS-DA), and a total of 52 metabolites in both the targeted and non-targeted metabolites were selected as biomarkers through VIP and ANOVA tests to construct a heatmap. Five metabolic pathways such as the pentose phosphate pathway (PPP), histidine metabolism, phenylalanine metabolism, alanine/aspartate/glutamate metabolism, and phenylalanine/tyrosine/tryptophan biosynthesis, were observed to show toxicologically significant alterations. Oxidative stress was also confirmed through MDA and ROS assays. Such perturbations of the metabolic pathways of zebrafish caused by the exposure to lindane resulted in significant toxicological effects.

Endosulfan sulfate is a major oxidized metabolite of endosulfan, which is a broad-spectrum chlorinated cyclodiene insecticide. In this study, GC-MS-based metabolic profiles of dead or live carp (Cyprinus carpio) exposed to endosulfan sulfate were investigated to elucidate the molecular toxicological effects of endosulfan sulfate on carp. Three different extraction methods were compared, and a 50% methanol solution was chosen as an efficient extraction method. Carp was exposed to endosulfan sulfate at a concentration of 8 ppb for 2 days. After exposure, the whole body of the fish was homogenized with liquid N 2 , extracted with the 50% methanol solution and dried before TMS derivatization for GC-MS analyses of the dead and live carp. A SIM (selected ion monitoring)-library of 373 metabolites was applied after GC-MS analysis to detect 146 metabolites in carp. Based on the one-way ANOVA results (P < 0.001) and fold changes of metabolites in dead carp versus control (fold change > 1.5 or < 0.667), 30 metabolites were identified as biomarkers that were significantly different in the metabolic profiles among the control, dead and live carp. A metabolic pathway analysis using MetaboAnalyst 4.0 revealed that those biomarkers were important for the living or death response to endosulfan sulfate. The pathways indicated by the metabolic pathway analysis included starch and sucrose metabolism, galactose metabolism, glycerolipid metabolism, the citrate cycle and linoleic acid metabolism. These results suggest that these pathways underwent significant perturbations over the exposure period.

X-ray crystallography is an important method used to confirm the three-dimensional structure of a chemical compound. In this study, the crystal structure of endosulfan sulfate was investigated. Endosulfan sulfate is the major metabolite of the insecticide endosulfan, which is composed of two stereoisomers (α and β). From GC-MS analysis, α-and β-endosulfan each gave a single peak in the endosulfan sample, but only one peak was observed for endosulfan sulfate. Interestingly, in X-ray crystallography, two conformers of endosulfan sulfate (A and B) were observed at a ratio of 2(A):1(B). A heterocyclic seven-membered ring of conformer B assumed a horizontal-chair form, differing from two twisted forms of α-endosulfan while a vertical-chair form was observed for conformer A, showing the very similar structure to β-endosulfan; this difference in conformation is caused by differing bond angles at O(1)-C(8)-C(3) and O(2)-C(9)-C(4). In space packing, two asymmetric units were obtained, and three molecules were aligned in the order of A-A-B conformers in each unit. The total potential energy of A was slightly lower (approximately 4 kcal/mol) than B, possibly resulting in the two molecules of A that exist in a rigid crystal state. However, A and B conformers should not exist at room temperature in a solution state for GC-MS analysis, likely due to the small energy difference. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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