The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP.
Background and purpose The role of CLOCK in regulation of drug chronoefficacy and chronotoxicity remains elusive. Here, we aimed to uncover the impact of CLOCK and dosing time on clopidogrel efficacy and toxicity. Experimental Approach The antiplatelet effect, toxicity and pharmacokinetics experiments were conducted with Clock −/− and wild-type mice after gavage administration of clopidogrel at different circadian time points. The expression levels of drug metabolizing enzymes were determined by qPCR and Western blotting. Transcriptional gene regulation was investigated using luciferase reporter and chromatin immunoprecipitation assays. Key Results The antiplatelet effect and toxicity of clopidogrel in wild-type mice showed a dosing-time dependent variation. Clock ablation reduced the antiplatelet effect of clopidogrel, increased clopidogrel-induced hepatotoxicity, and blunted their rhythms. This was associated with attenuated rhythms of clopidogrel active metabolite (Clop-AM) and clopidogrel, respectively. We further found that Clock regulated the diurnal variation of Clop-AM formation by modulating the rhythmic expression of CYP1A2 and CYP3A11, and altered clopidogrel chronopharmacokinetics by circadian regulation of CES1D expression. Mechanistic studies revealed that CLOCK activated Cyp1a2 and Ces1d transcription by directly binding to the E-box elements in their promoters, and promoted Cyp3a11 transcription through enhancing the transactivation activity of DBP and TEF. Conclusion and implication CLOCK regulates the diurnal rhythmicity in clopidogrel efficacy and toxicity through regulation of CYP1A2, CYP3A11 and CES1D expression. The findings may contribute to the optimization of dosing schedule for clopidogrel, and deepen understanding of the connection between the circadian clock and chronopharmacology.
Type 2 diabetes mellitus (T2D) is a complex metabolic disorder requiring polypharmacy treatment in clinic, with metformin being widely used antihyperglycemic drug. However, the mechanisms of metformin as a perpetrator inducing potential drug-drug interactions and adverse drug reactions are scarcely known to date. Carboxylesterases (CESs) are major hydrolytic enzymes highly expressed in the liver, including mouse carboxylesterase 1d (Ces1d) and Ces1e. In the present study, experiments are designed to investigate the effects and mechanisms of metformin on Ces1d and Ces1e in vivo and in vitro. In results, metformin suppresses the expression and activity of Ces1d and Ces1e in a dose- and time-dependent manner. The decreased expression of nuclear receptor PXR and its target gene P-gp indicates the involvements of PXR in the suppressed expression of carboxylesterases by metformin. Furthermore, metformin significantly suppresses the phosphorylation of AMPK and JNK, and the suppression of carboxylesterases induced by metformin is repeatedly abolished by AMPK inhibitor Compound C and JNK inhibitor SP600125 . It implies that the activation of AMPK and JNK pathways mediates the suppression of carboxylesterases by metformin. The findings deserve further elucidation including clinical trials and have a potential to make contribution for the rational medication in the treatment of T2D patients.
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