Dexamethasone transcriptionally increases the expression of the pregnane X receptor and synergistically enhances pyrethroid esfenvalerate in the induction of cytochrome P450 3A23

The pregnane X receptor (PXR) is recognized as a key regulator for the induction of a large number of genes in drug metabolism and transport. The transactivation of PXR is enhanced by the glucocorticoid dexamethasone and the enhancement is linked to the induction of PXR in humans and rats. The present study was undertaken to determine the mechanism for the induction and ascertain the synergistic effect on the expression of CYP3A23, a rat PXR target. In primary hepatocytes, significant induction of PXR was detected as early as 2 h after the treatment and the maximal induction occurred at 1 μM dexamethasone. Similar induction kinetics was observed in the hepatoma line H4-II-E-C3. The induction was abolished by actinomycin D and dexamethasone efficaciously stimulated the rat PXR promoter. In addition, dexamethasone synergized esfenvalerate (an insecticide and a PXR activator) in inducing CYP3A23 and stimulating the CYP3A23 promoter. The full promoter of CYP3A23 (−1445/+74) was activated in a similar pattern as the changes in PXR mRNA in response to dexamethasone, esfenvalerate and co-treatment. In contrast, different responding patterns were detected on the stimulation of the CYP3A23 proximal promoter. Synergistic stimulation was also observed on the CYP3A4-DP-Luc reporter, the human counterpart of CYP3A23. These findings establish that transactivation is responsible for the induction of rat PXR and the induction presents potential interactions with insecticides in a species-conserved manner. The different responding patterns among CYP3A23 reporters point to an involvement of multiple transcriptional events in the regulation of CYP3A23 expression by dexamethasone, esfenvalerate and both. [Refer to PDF for graphical abstract

Dexamethasone suppresses the expression of multiple rat carboxylesterases through transcriptional repression: Evidence for an involvement of the glucocorticoid receptor

Carboxylesterases play important roles in the metabolism of xenobiotics and detoxication of insecticides. Without exception, all mammalian species studied express multiple forms of carboxylesterases. Several rat carboxylesterases are well-characterized including hydrolase A, B and S, and the expression of these enzymes is significantly suppressed by glucocorticoid dexamethasone. In this study, we used multiple experimental systems and presented a molecular mechanism for the suppression. Rats receiving one or more daily injections of dexamethasone consistently expressed lower HA, HB and HS. The suppression occurred at the levels of mRNA, protein and hydrolytic activity. In hepatoma cell line H4-II-E-C3, nanomolar dexamethasone caused significant decreases in HA, HB and HS mRNA, and the decreases were abolished by antiglucocorticoid RU486. Additionally, dexamethasone at nanomolar concentrations repressed the promoters of carboxylesterases, and the repression was reduced by glucocorticoid receptor-β, a dominant negative regulator of the glucocorticoid receptor (GR). In contrast, co-transfection of the pregnane X receptor (PXR) increased the reporter activities, but the increase occurred only at micromolar concentrations of dexamethasone. These findings establish that both GR and PXR are involved in the regulated expression of rat carboxylesterases by dexamethasone but their involvement depends on the concentrations

Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2

Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health

Surge in Expression of Carboxylesterase 1 During the Post-neonatal Stage Enables a Rapid Gain of the Capacity to Activate the Anti-influenza Prodrug Oseltamivir

Background. Oseltamivir, a widely used anti-influenza drug, is hydrolytically activated by carboxylesterase 1 (CES1). The expression of this carboxylesterase is developmentally regulated. This study was performed to determine when after birth infants acquire competence of activating this prodrug. Methods. Liver tissue samples were collected and divided into 5 age groups: group 1 (1-31 d old), group 2 (35-70 d old), group 3 (89-119 d old), group 4 (123-198 d old), and group 5 (>18 years of age). These samples were analyzed for oseltamivir hydrolysis and CES1 expression. Results. Liver samples in group 1 expressed the lowest level of CES1 with the lowest hydrolytic activity toward oseltamivir. A 4-7-fold increase between groups 1 and 2 (1-31 vs 35-70 d of age) was detected in the hydrolysis and expression analyses, respectively. Liver samples in the other 3 pediatric groups (35-198 d of age) exhibited similar expression and hydrolysis levels. Overall, liver samples in group 1 had CES1 expression and hydrolysis levels that were 10% of those of adults, whereas liver samples in the other 3 pediatric groups had levels that were ∼50% of adult levels. Conclusions. The post-neonatal surge in CES1 expression ensures the hydrolytic capacity to be gained rapidly after birth in infants, but the larger variability during this period suggests that caution should be exercised on the extrapolated dosing regimens of ester drugs from other age group

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses

Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes

Hypolipidemic agent Z-guggulsterone: metabolism interplays with induction of carboxylesterase and bile salt export pump

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens

Online bin packing with arbitrary release times

AbstractWe study a new variant of the online bin-packing problem, in which each item ai is associated with a size ai and also a release time ri so that it must be placed at least ri above the bottom of a bin. Items arrive in turn and must be assigned without any knowledge of subsequent items. The goal is to pack all items into unit-size bins using the minimum number of bins. We study the problem with all items have equal size. First, we show that the ANY FIT algorithm cannot be approximated within any constant. Then we present a best possible online algorithm with asymptotic competitive ratio of two

Scoparone potentiates transactivation of the bile salt export pump gene and this effect is enhanced by cytochrome P450 metabolism but abolished by a PKC inhibitor

Background and Purpose Hyperbilirubinaemia and cholestasis are two major forms of liver abnormality. The Chinese herb Yin Chin has been used for thousands of years to treat liver dysfunctions. In mice, this herb and its principal ingredient scoparone were found to accelerate the clearance of bilirubin accompanied by the induction of uridine diphosphate-5′- glucuronosyltransferase-1A1 (UGT1A1), a bilirubin processing enzyme. The aim of this study was to determine whether scoparone induces the expression of human UGT1A1. In addition, the expression of the bile salt export pump (BSEP), a transporter of bile acids, was determined. Experimental Approach Primary human hepatocytes and hepatoma line Huh7 were treated with scoparone, chenodeoxycholic acid (CDCA) or both. The expression of UGT1A1 and BSEP mRNA was determined. The activation of the human BSEP promoter reporter by scoparone was determined in Huh7 cells by transient transfection and in mice by bioluminescent imaging. The metabolism of scoparone was investigated by recombinant CYP enzymes and pooled human liver microsomes. Key Results Scoparone did not enhance the expression of either human BSEP or, surprisingly, UGT1A1. However, scoparone significantly potentiated the expression of BSEP induced by CDCA. Consistent with this, scoparone potentiated the stimulant effect of CDCA on the human BSEP promoter. This potentiation was enhanced by co-transfection of cytochrome P4501A2 but abolished by the PKC inhibitor GF109203X. ConclusionS and Implications Scoparone and Yin Chin normalize liver function primarily by enhancing the secretion of bile acids, and this effect probably varies depending on the metabolic rate of scoparone. © 2011 The British Pharmacological Society