Carboxylesterase Activity Research Articles

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer’s disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure–activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced β-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Glyphosate and glufosinate ammonium, herbicides commonly used on genetically modified crops, and their interaction with microplastics: Ecotoxicity in anuran tadpoles

The effects of glyphosate (GLY)-based and glufosinate ammonium (GA)-based herbicides (GBH and GABH, respectively) and polyethylene microplastic particles (PEMPs) on Scinax squalirostris tadpoles were assessed. Tadpoles were exposed to nominal concentrations of both herbicides (from 1.56 to 100 mg L−1) and PEMPs (60 mg L−1), either alone or in combination, and toxicity evaluated at 48 h. Acetylcholinesterase (AChE), carboxylesterase (CbE), and glutathione-S-transferase (GST) activities were analyzed at the three lowest concentrations (1.56, 3.12 and 6.25 mg L−1, survival rates >85%) of both herbicides alone and with PEMPs. Additionally, the thermochemistry of the interactions between the herbicides and polyethylene (PE) was analyzed by Density Functional Theory (DFT). The median-lethal concentration (LC50) was 43.53 mg L−1 for GBH, 38.56 mg L−1 for GBH + PEMPs, 7.69 for GABH, and 6.25 mg L−1 for GABH+PEMPs. The PEMP treatment increased GST but decreased CbE activity, whereas GBH and GABH treatments increased GST but decreased AChE activity. In general, the mixture of herbicides with PEMPs increased the effect observed in the individual treatments: the highest concentration of GBH + PEMPs increased GST activity, whereas GABH+PEMP treatments decreased both AChE and CbE activities. DFT analysis revealed spontaneous interactions between the herbicides and PE, leading to the formation of bonds at the herbicide-PE interface, significantly stronger for GA than for GLY. The experimental and theoretical findings of our study indicate that these interactions may lead to an increase in toxicity when pollutants are together, meaning potential environmental risk of these combinations, especially in the case of GA.

Evaluation of teratogenic and toxic effects of enrofloxacin-based antibiotic on zebrafish (Danio rerio) larvae with biochemical and developmental markers

ABSTRACT In this study, the effects of enrofloxacin-based antibiotic (EBA) used as a drug in veterinary medicine were evaluated on zebrafish embryos and larvae. The embryos were exposed to 17.56–300 mg L − 1 EBA for 96 h and survival rates and body malformations were evaluated under a stereo-microscope. In addition, 10-days larvae were exposed to LC50/4, LC50/2 and LC50 concentrations of antibiotic for 72 h and activities of glutathione S-transferase (GST), glutathione reductase, carboxylesterase, acetylcholinesterase, lactate dehydrogenase (LDH) and aspartate aminotransferases (AST) were measured. LC50 and EC50 values and the teratogenic index (TI) were calculated as 150.1 and 124.8 mg L − 1 and 1.2, respectively. EBA was teratogen for zebrafish embryos according to calculated TI. Spinal curvature, tail malformation, pericardial oedema and yolk sac oedema were determined in embryos exposed to 88.8 mg L − 1 and higher EBA concentrations. Furthermore, significant growth inhibitions were determined in larvae exposed to 59.26 mg L − 1 and above EBA concentrations. Also, 75 and 150 mg L − 1 concentrations of EBA caused a significant increase in GST, LDH and AST activities in zebrafish larvae. These results show that EBA causes adverse effects on zebrafish development and may adversely affect the aquatic ecosystem if it enters the aquatic environment.

Chemical and Biological Assay for The Degradation of Microwave Synthesized-Hyperbranched Poly (Urethane-Urea) By Local Bacterial Isolates

Ultra-fast recently Microwave-synthesized Hyperbranched Poly (urethane-urea) (MW-HBPU), is a polymer with a unique morphological structure and different applications in many fields as textile printing and coating. Because Polyurethanes (PUs) have excellent chemical and physical properties, their bio-decomposition and decay are difficult and time consuming. Therefore, MW-HBPU biodegradation by five bacterial isolates isolated from different local garbage sites has been studied. The highest activity was observed in three isolates (H1, H2 and H3), that were identified by morphology, physiology, biochemical and molecular biology (16S rRNA). Achromobacter sp. strain NRC2018, Bacillus subtilis strain NRC2018 and Bordetella petrii strain NRC2018, respectively. By determination of Carboxylesterase and acetylcholineesterase activity for the three strains. Achromobacter sp. strain NRC 2018 showed high activity give 91.2 and 526.4 U/ml for Carboxylesterase and acetylcholineesterase, respectively. The change in the characteristic functional groups reflected in the ATR-FTIR spectra of MW-HBPU after incubation with these bacteria showed attack at certain sites in the MW-HBPU structure and pointed to structural changes. SEM images clearly showed degradation in the MW-HBPU network. Additionally, a notable change in weight after incubation with isolated bacteria was also used to demonstrate the ability of these strains to effectively biodegrade MW-HBPU.

Chemical composition of essential oils from Thymus mongolicus, Cinnamomum verum, and Origanum vulgare and their acaricidal effects on Haemaphysalis longicornis (Acari: Ixodidae)

Chemical acaricides are mainly used in traditional tick control, which leads to the emergence of tick resistance and concurrently results in environmental pollution. In the present study, the chemical constituents of essential oils (EOs) from Thymus mongolicus, Cinnamomum verum, and Origanum vulgare was analyzed, and their potential application was evaluated to control the vector tick Haemaphysalis longicornis, which is widely distributed over vast areas of Eurasia, Australia, and New Zealand. Gas chromatography-mass spectrometry analysis revealed that the phenols thymol and carvacrol accounted for 34.66% and 75.72% of the EOs of T. mongolicus and O. vulgare, respectively, whereas trans-cinnamaldehyde (49.42%) was the main constituent of C. verum EO. Immersion tests showed that the EOs of C. verum and O. vulgare had significant acaricidal activity against larval H. longicornis, with the 50% lethal concentration (LC50) being 16.07 and 18.02 mg/mL, respectively, and the 95% lethal concentration (LC95) being 120.37 and 130.09 mg/mL, respectively. The EOs of O. vulgare and T. mongolicus showed significant acaricidal activity against unfed adult H. longicornis, with LC50 being 43.50 and 44.21 mg/mL, respectively, and LC95 being 113.66 and 137.99 mg/mL, respectively. The fumigant toxicity test showed significant acaricidal activity of the three EOs against both unfed and engorged nymphal and adult H. longicornis. Enzyme assays revealed that the EOs of both C. verum and O. vulgare significantly inhibited glutathione S-transferase activity (P < 0.05). In contrast, the activities of carboxylesterase and multifunction oxidases were significantly inhibited by EOs extracted from all three plants (P < 0.05). Taken together, these findings suggest that plant EOs may serve as an environment-friendly alternative for synthetic acaricides in future tick control.

Enzymatic responses in the head and midgut of Africanized Apis mellifera contaminated with a sublethal concentration of thiamethoxam

The increasing use of insecticides, promoted by the intensification of agriculture, has raised concerns about their influence on the decline of bee colonies, which play a fundamental role in pollination. Thus, it is fundamental to elucidate the effects of insecticides on bees. This study investigated the damage caused by a sublethal concentration of thiamethoxam - TMX (0.0227 ng/μL of feed) in the head and midgut of Africanized Apis mellifera, by analyzing the enzymatic biomarkers, oxidative stress, and occurrence of lipid peroxidation. The data showed that the insecticide increased acetylcholinesterase activity (AChE) and glutathione-S-transferase (GST), whereas carboxylesterase (CaE3) activity decreased in the heads. Our results indicate that the antioxidant enzymes were less active in the head because only glutathione peroxidase (GPX) showed alterations. In the midgut, there were no alkaline phosphatase (ALP) or superoxide dismutase (SOD) responses and a decrease in the activity of CaE was observed. Otherwise, there was an increase in GPX, and the TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The results showed enzymes such as CaE3, GST, AChE, ALP, SOD, and GPX, as well as the TBARS assay, are useful biomarkers on bees. They may be used in combination as a promising tool for characterizing bee exposure to insecticides.

Sensitivity of midgut physiological parameters of Lymantria dispar L. larvae to benzo[a]pyrene in populations with different multigeneration contact to environmental pollutants

Accumulation of organic pollutants in the environment calls for sensing physiological parameters adequate to indicate the presence of contaminants and their effects on ecosystems. Evidence points to the importance of insect adaptations in their habitats for the assessment of sensitive biomarkers so we examined the influence of origin and multigenerational adaptations of the Lymantria dispar larvae to chronic benzo[a]pyrene (B[a]P) treatment under laboratory conditions. The main aim was to compare reactions of larvae from unpolluted and polluted forests using alkaline phosphatase (ALP), acid phosphatase (ACP), and carboxylesterase (CE) specific activities in the midgut, including electrophoretic isoform patterns; midgut expression levels of Hsp70, larval development time (DT), and midgut mass (MM), after chronic exposure to 5 and 50 ng of B[a]P/g dry food weight. The biomarker potential of these parameters regarding larval pre-exposure history to pollution was estimated by principal component analysis (PCA). B[a]P treatment resulted in inhibition of ALP activity, a rise of CE activity, and reduction of MM in larvae from the unpolluted forest, while the population from the polluted forest showed significant elevation of Hsp70 expression in the midgut, prolonged DT, and reduction of MM. PCA confirmed variations in responses of the selected parameters regarding population origin. The obtained results provide insight into insect population variability concerning physiological responses to pollutants. It is indicative that all investigated physiological parameters of L. dispar larvae showed origin-dependent responses to long-term presence of B[a]P, which may be of great importance in ecotoxicological research.

Enzyme Activities as Indicators of Soil Quality: Response to Intensive Soybean and Rice Crops

Soil enzyme activities are often used as indicators of soil contamination. The responses of the activities of specific soil enzymes, dehydrogenase, acid phosphatase, β-glucosidase, carboxylesterase, and urease to different land uses (soybean and rice crops, and a reference site) were analyzed. Changes in activity at the start and end of each crop cycle were quantified. In general, the catalytic activity of all enzymes was lower in both crops than in the reference site. Regarding the soybean crop, all the enzyme activities decreased at the start of the crop cycle (27.5–53%, with respect to reference site values), whereas only acid phosphatase, β-glucosidase and carboxylesterase were lower at the end of the cycle (70.3%, 29.44%, and 45.79%; respectively). In the rice crop, dehydrogenase, acid phosphatase, and β-glucosidase activities were lower at the start of the cycle (27.88%, 50.32%, and 23.21%; respectively), with respect to reference site. However, the enzyme activity was lower at the end of the cycle compared to the reference site (dehydrogenase 20.47%, acid phosphatase 72.72%, β-glucosidase 57.77%, carboxylesterase 27.59%), except for urease activity. Current results suggested that the use of enzyme activities as indicators of soil quality is a viable approach to assess the pesticide impact in agricultural soils of Argentina.

Rational Design of a Highly Selective Near-Infrared Two-Photon Fluorogenic Probe for Imaging Orthotopic Hepatocellular Carcinoma Chemotherapy.

Selective fluorescence imaging of biomarkers in vivo and in situ for evaluating orthotopic hepatocellular carcinoma (HCC) chemotherapy remains a great challenge due to current imaging agents suffering from the potential interferences of other hydrolases. Herein, we observed that carbamate unit showed a high selectivity toward the HCC-related biomarker carboxylesterase (CE) for evaluation of treatment. A near-infrared two-photon fluorescent probe was developed to not only specially image CE activity in vivo and in situ but also target orthotopic liver tumor after systemic administration. The in vivo signals of the probe correlate well with tumor apoptosis, making it possible to evaluate the status of treatment. The probe enables the imaging of CE activity in situ with a high-resolution three-dimensional view for the first time. This study may promote advances in optical imaging approaches for precise imaging-guided diagnosis of HCC in situ and its evaluation of treatment.

Rational Design of a Highly Selective Near‐Infrared Two‐Photon Fluorogenic Probe for Imaging Orthotopic Hepatocellular Carcinoma Chemotherapy

AbstractSelective fluorescence imaging of biomarkers in vivo and in situ for evaluating orthotopic hepatocellular carcinoma (HCC) chemotherapy remains a great challenge due to current imaging agents suffering from the potential interferences of other hydrolases. Herein, we observed that carbamate unit showed a high selectivity toward the HCC‐related biomarker carboxylesterase (CE) for evaluation of treatment. A near‐infrared two‐photon fluorescent probe was developed to not only specially image CE activity in vivo and in situ but also target orthotopic liver tumor after systemic administration. The in vivo signals of the probe correlate well with tumor apoptosis, making it possible to evaluate the status of treatment. The probe enables the imaging of CE activity in situ with a high‐resolution three‐dimensional view for the first time. This study may promote advances in optical imaging approaches for precise imaging‐guided diagnosis of HCC in situ and its evaluation of treatment.

A Comparative Studies of the Activities of Phenoloxidase, Carboxylesterase and Alkaline Phosphatase from Different Geographic Strains of Flacherie-Infected Bombyx mori

A Comparative Studies of the Activities of Phenoloxidase, Carboxylesterase and Alkaline Phosphatase from Different Geographic Strains of Flacherie-Infected Bombyx mori

Characterization of Ocimum basilicum synthesized silver nanoparticles and its relative toxicity to some insecticides against tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae)

Tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae) is a notorious cosmopolitan pest of numerous crops. Frequent evolution of insecticide resistance and polyphagous nature favors the survival of this voracious pest. Nanotechnology offers an alternative technique to overcome the limitations of existing pest management strategies. In the present study, the silver nanoparticles were synthesized from Ocimum basilicum leaf extract and UV-spectra at 464.17 nm confirmed their synthesis while crystalline nature of nanoparticles was evaluated by X-rays diffraction (XRD) pattern and scanning electron microscopy (SEM) revealed their octagonal to spherical shape. Insecticidal potential of O. basilicum synthesized silver nanoparticles (ObAgNPs) was evaluated in comparison with some synthetic insecticides owning different mode of actions, including Coragen®, Proclaim®, Tracer® and Talstar® against S. litura. Probit analysis of 2nd, 3rd, 4th and 5th instars of S. litura showed that lethal concentrations (LCs) of tested nanoparticles and pesticides were positively correlated with older instars. All values of LC50, LC90 and LC95 were statistically different from each other with maximum on the 4th instar and minimum on 2nd instar of S. litura. Tukey’s HSD test revealed that ObAgNPs caused maximum mortality (21.67–96.67%) in 2nd instar larvae at the rate of 100–1500 mg/L followed by Coragen® (18.33–91.67%), Proclaim® (13.33–78.33%) and Talstar® (13.33–68.33%), while Tracer indicated the lowest larval mortality (11.67–66.67%) at the rate of 60–120 mg/L and a similar trend was assessed for all the successive instars of S. litura. Higher activity of carboxyl esterase and glutathione-s-transferases confirmed the development of insecticide resistance against the synthetic chemicals. Conclusively, ObAgNPs were found to be eco-friendly but the most effective biogenic compounds for the suppression of S. litura population as compared to the selected synthetic chemicals which may be hazardous for the environment as well as human health. This study will assist the pesticide industries to re-standardize the dose rates against Spodoptera litura.

Evaluating effects of black carrot extract on testicular carboxylesterase activity and oxidative stress parameters in rats exposed to Bisphenol A

We aimed to evaluate the effects of black carrot extract on testicular carboxylesterase (Ces) activity, malondialdehyde (MDA), glutathione S-transferases (GST) and reduced glutathione (GSH) in male rats exposed to bisphenol A (BPA). Adult Wistar albino male rats were divided into 4 groups as follows (n = 7/group): control, BPA, black carrot and BPA+Black carrot. Testicular Ces, MDA, GST and reduced GSH were analyzed by using a spectrophotometer system. Testicular Ces activity was significantly lower only in the BPA group than the control group (p < 0.001). Testicular MDA concentrations were higher only in the BPA group compared with the control group (p < 0.001). Reduced GSH level was higher in the black carrot and BPA+Black carrot groups than the control group (p < 0.05). GST activity was lower in the BPA group in comparison to the control group (p < 0.05). In the black carrot and BPA+Black carrot groups, the GST activities were higher than the control group (p < 0.001). Our results showed BPA suppresses testicular detoxification activity and increases lipid peroxidation in rats. We observed that black carrot extract has a beneficial effect on the toxic and oxidative stress-related parameters caused by BPA exposure.

Carboxylesterases and arylacetamide deacetylase comparison in human A549, H460, and H727 pulmonary cells

AimsHuman carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions. Materials and methodsThe p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes. Key findingsAADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar Km values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the pNPA hydrolysis by S9 fractions were significantly lower than those detected in living cells. Inhibition studies revealed that 4-MUA hydrolysis was inhibited by bis-p-nitrophenyl phosphate and phenylmethanesulfonyl fluoride more than loperamide; on the contrary, pNPA hydrolysis inhibition was limited with similar inhibition profiles being obtained in both living cells and S9 fractions. The presence of carboxylesterases was definitely confirmed by cellular bioimaging. SignificanceThese findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.

Carboxylesterase Notum Is a Druggable Target to Modulate Wnt Signaling.

Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling.

Biomarker responses and metabolism in Lumbricus terrestris exposed to drugs of environmental concern, an in vivo and in vitro approach

The earthworm Lumbricus terrestris is an anecic species living in natural soils but it is also a sentinel in pollution monitoring. Specimens of L.terrestris were exposed for 48 h though the filter paper contact test at 1 mg/mL of the chemicals: Lamotrigine (LMG), Cocaine (COC), Fipronil (FIP) and the pesticide bis-4-nitrophenyl phosphate (BNPP). After that period, the activities of Acetylcholinesterase, Glutathione S-transferase, Carboxylesterase (CE) using different substrates, and lipid peroxidation levels were evaluated in the exposed whole tissue earthworms. The results revealed differences only in CE activity, with 4-nitrophenyl butyrate (4NPB) and 1-naphthyl butyrate (1NB) the most responsive substrates to COC. The kinetic parameters of CE were characterized, for the first time, in whole tissue of this species. The chemical analysis by LC-MS/MS, confirmed the exposure to the parent compounds, identified metabolites and evidenced biotransformation pathways in earthworms. Metabolic reactions included oxidation (LMG and FIP), hydrolysis (COC and FIP) as well as glycosylation (LMG, COC and FIP). A hitherto unknown metabolite of LMG due to the conjugation with phenylalanine glutamine was formed. The in vivo results on CE activity with the specific inhibitor, BNPP, were confirmed in vitro. Moreover, in the in vitro approach, the inclusion of other contaminants of environmental concern supports the potential of CE as biomarker. This study identifies the main metabolites formed by earthworms for further in vivo exposures under more realistic conditions and the potential use of CE measures as biomarker of emerging contaminants.

Effects of Inoculants Producing Antifungal and Carboxylesterase Activities on Corn Silage and Its Shelf Life against Mold Contamination at Feed-Out Phase.

The present study aimed to investigate effects of dual-purpose inoculants (antifungal and carboxylesterase activities) not only on corn silage quality, but also its shelf life against mold contamination at feed-out phase. Corn forage was ensiled for 252 d with different inoculants of the following: control (CON), Lactobacillus brevis 5M2 (5M), Lactobacillus buchneri 6M1 (6M), and mixture of 5M and 6M at 1:1 ratio (MIX). After ensiling, corn silage was contaminated with Fusarium graminearum. Silages applied inoculants had positive effects by increased organic acid and lactic acid bacteria, and decreased undesirable microbes. At feed-out phase, contamination of F. graminearum into corn silage had a negative effect on aerobic stability caused by increased growth of undesirable microbes. However, silages applied inoculants had positive effects by decreased undesirable microbes and extended lactic acid bacteria and aerobic stability. Generally, MIX silage presented better effects on organic acid production, rumen degradation, inhibition of undesirable microbes, and aerobic stability than 5M silage and 6M silage. The present study concluded that application of inoculants into corn silage had positive effects on fermentation characteristics and extended shelf life against mold contamination at feed-out phase. A mixed inoculant appeared to have better effects of antifungal and carboxylesterase than a single inoculant.

Biomarkers in rock oysters (Saccostrea mordax) in response to organophosphate pesticides

Chlorpyrifos is a xenobiotics contaminants that threats the marine environment and the living organism within the habitat. Although several marine bivalve species have been used as the indicator of marine pollution, the used of Saccostrea mordax is remaining unknown. This study aimed at investigating the suitability of lysosome membrane integrity, carboxylesterase activity, 8-oxo-2′-deoxyguanosine and condition index as biomarkers in adult S. mordax following their exposure to 0.0, 5.0 and 500 μg.L-1 of Chlorpyrifos for 21 days under laboratory conditions. Results indicated that the lysosome membrane integrity showed a dose-dependent response with a significant statistical number of destabilized cells between all the treatment groups. Carboxylesterase activity was significantly inhibited in 500 μg.L-1 chlorpyrifos treated group, while the environmentally relevant concentration (5 μg.L-1) did not induce a significant inhibition with reference to the control. Similarly, the condition index showed a dose-dependent response with the oysters exposed to 500 μg.L-1 chlorpyrifos exhibiting a significantly reduced growth rate. There was no statistical significance in the means of both 8-oxo-2′-deoxyguanosine in all treatment groups. The reaction of S. mordax to chlorpyrifos contamination demonstrates that the species can potentially be used as sentinel organisms in environmental monitoring programs. Lysosome membrane integrity was a single out as a sensitive biomarker for exposure to chlorpyrifos and is therefore suitable for environmental monitoring for sublethal concentrations of chlorpyrifos contaminations. Additionally, the use of multiple biomarkers was found to be robust in this study and can be extrapolated to other ecotoxicological studies

Effect of UGT1A1, CYP3A and CES Activities on the Pharmacokinetics of Irinotecan and its Metabolites in Patients with UGT1A1 Gene Polymorphisms.

Irinotecan (CPT-11) is metabolized to an active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterase (CES). SN-38 is then converted to the inactive metabolite SN-38 glucuronide (SN-38G) by glucuronosyltransferase 1A1 (UGT1A1). Genetic polymorphisms in UGT1A1 have been associated with altered SN-38 pharmacokinetics, which increase the risk of toxicity in patients. CPT-11 is also converted to 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC) by cytochrome P450 3A (CYP3A), and this route also affects the plasma concentration of SN-38. We evaluated the activities of UGT1A1, CYP3A, and CES and the factors affecting the pharmacokinetics of plasma SN-38 in patients with UGT1A1 gene polymorphisms. Three male patients aged 56, 65, and 49 years were recruited for the analysis. All patients had pancreatic cancer, received FOLFIRINOX, and had UGT1A1*6/*6 (patients 1 and 3) or *6/*28 (patient 2) genetic polymorphisms. The rate constants for evaluating the enzyme activity were determined from the measured plasma concentration of CPT-11 and its metabolites using a two-compartment model by WinNonlin. The area under the plasma concentration-time curve (AUC) of SN-38 was patient 1 > patient 2 > patient 3. The rate constants obtained from the model analysis indicated the respective enzyme activities of UGT1A1 (k57), CYP3A (k13 + k19), and CES (k15). The order of values for UGT1A1 activity was patient 2 > patient 3 > patient 1. Since UGT1A1 activity was low in patient 1 with a high AUC of SN-38, it can be said that the increase in plasma concentration was due to a decrease in UGT1A1 activity. Conversely, the order of values for CYP3A and CES activities was patient 3 > patient 1 > patient 2 and patient 2 > patient 1 > patient 3, respectively. Patient 3 had the lowest AUC of SN-38, caused by a lower level of CES activity and increased CYP3A activity. In this study, we indicated that the plasma AUC of SN-38 and AUC ratio of SN-38G/SN-38 may depend on changes in the activities of CYP3A, CES, and UGT1A1. Using pharmacokinetic analysis, it is possible to directly evaluate enzyme activity and consider what kind of enzyme variation causes the increase in the AUC of SN-38.

Clock gene Bmal1 controls diurnal rhythms in expression and activity of intestinal carboxylesterase 1.

We aimed to characterize diurnal rhythms in CES1 expression and activity in mouse intestine, and to investigate a potential role of the core clock gene Bmal1 in generating diurnal rhythms. The regulatory effects of intestinal Bmal1 on diurnal CES1 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Metabolic activity of CES1 in vitro and in vivo were determined by microsomal assays and pharmacokinetic studies, respectively. Transcriptional gene regulation was investigated using luciferase reporter assay. Total CES1 protein varied significantly according to time of the day in wild-type (Bmal1fl/fl) mice, peaking at ZT6. Of detectable Ces1 genes, Ces1d mRNA displayed a robust diurnal rhythm with a peak level at ZT6, whereas mRNAs of Ces1e, 1f and 1g showed no rhythms in wild-type mice. Loss of intestinal Bmal1 reduced the levels of total CES1 protein and Ces1d mRNA, and blunted their diurnal rhythms in mice. In vitro microsomal assays indicated that intestinal metabolism of mycophenolate mofetil (MMF, a known CES1 substrate) was more extensive at ZT6 than at ZT18. ZT6 dosing of MMF to wild-type mice generated a higher systemic exposure of mycophenolic acid (the active metabolite of MMF) as compared with ZT18 dosing. Intestinal ablation of Bmal1 down-regulated CES1 metabolism at ZT6, and abolished its time-dependency both in vitro and in vivo. Furthermore, Ces1d/CES1 rhythmicity and positive regulation of Ces1d/CES1 by BMAL1 were confirmed in CT26 and Caco-2 cells. Mechanistically, BMAL1 trans-activated Ces1d/CES1 probably via binding to the E-box elements in the gene promoters. Bmal1 controls diurnal rhythms in expression and activity of intestinal CES1. Our findings have implications for understanding the crosstalk between circadian clock and xenobiotic metabolism in the intestine.