Juvenile Hormone (JH) Esterase of the Mosquito Culex quinquefasciatus Is Not a Target of the JH Analog Insecticide Methoprene

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (kcat/KM ratio) and Vmax values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE

Induction of Amyloid-beta(42) Production by Fipronil and Other Pyrazole Insecticides

Generation of amyloid-β peptides (Aβs) by proteolytic cleavage of the amyloid-β protein precursor (AβPP), especially increased production of Aβ42/Aβ43 over Aβ40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer’s disease (AD). In familial AD (FAD), altered Aβ production originates from specific mutations of AβPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aβs remains unknown. We hypothesize that the ‘human chemical exposome’ contains products able to favor the production of Aβ42/Aβ43 over Aβ40 and shorter Aβs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aβ42/Aβ43 production. Nine pyrazole insecticides were found to induce a β- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aβ42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aβs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AβPP by highly purified γ-secretase toward Aβ42/Aβ43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aβ42/Aβ43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aβ42/Aβ43 peptides, suggesting the possible existence of environmental “Alzheimerogens” which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD

Triclocarban Mediates Induction of Xenobiotic Metabolism through Activation of the Constitutive Androstane Receptor and the Estrogen Receptor Alpha

Triclocarban (3,4,4′-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ERα) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car−/− mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human health effects from exposure to TCC

Effect of ionic liquids on epoxide hydrolase-catalyzed synthesis of chiral 1,2-diols

Ionic liquids (ILs) offer new possibilities for epoxide hydrolase (EH) catalyzed resolution of epoxides and for synthesis of chiral 1,2-diols. Soluble EHs from cress and mouse (csEH and msEH) and microsomal EH from rat (rmEH) were tested in several ILs. For all the enzymes tested, higher enantioselectivities were obtained in [bmim][N(Tf)(2)] and [bmim][PF(6)]. The optimized amount of water for EH activity in these ILs was established. Classical problems arising from low solubility of epoxides in water or from the high tendency of the oxirane ring to undergo chemical hydrolysis were avoided using these new media

A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure

There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments

Evidence of quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of murine Clara cells after exposure to naphthalene

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro

Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-beta-methylstyrene oxide catalyzed by soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) was shown to catalyze hydrolysis of epoxides using the ionic liquids (ILs) [bmim][PF(6)], [bmim][N(Tf)(2)], and [bmim][BF(4)] (where bmim = 1-butyl-3-methylimidazolium, PF(6) = hexafluorophosphate, N(Tf)(2) = bis(trifluoromethylsulfonyl)imide, and BF(4) = tetrafluoroborate) as reaction medium. Reaction rates were generally comparable with those observed in buffer solution, and when the cress enzyme was used the hydrolysis of trans-beta-methylstyrene oxide gave, through a stereoconvergent process, the corresponding optically active (1S,2R)-erythro-1-phenylpropane-1,2-diol. (C) 2004 Elsevier B.V. All rights reserved

1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase inhibitor, lowers L-NAME-induced hypertension through suppression of angiotensin-converting enzyme in rats.

ObjectiveThis study evaluated the efficacy of the soluble epoxide hydrolase (sEH) inhibitor, TPPU on chronic NG-Nitro L-arginine methyl ester (L-NAME)-induced hypertension in rats and its effects on plasma Angiotensin II (Ang II), cardiac Angiotensin-converting enzyme (ACE) and Angiotensin II receptor type 1 (AT1R) expressions.Materials and methodsForty Sprague Dawley rats were divided into 5 groups. Two groups served as control and received orally either vehicle or TPPU (3 mg/kg) for five weeks. The other three groups were given L-NAME (50 mg/kg/day) in drinking water for five weeks. Two weeks after the L-NAME treatment, animals received orally either saline or TPPU (3 mg/kg/day) or lisinopril (10 mg/kg/day) daily for 3 weeks. Blood pressure (BP) was measured weekly. At the end of the experiment, plasma Ang II, cardiac ACE and AT1R protein and gene expressions were determined.ResultsL-NAME caused a significant increase in BP of the animals. TPPU and lisinopril resulted in normalization of L-NAME-induced hypertension. They also caused a significant reduction in Ang II and ACE protein and gene expressions compared to L-NAME and vehicle-treated animals.ConclusionsThis study demonstrates that TPPU effectively lowers L-NAME-induced hypertension in rats. The mechanism of its antihypertensive effect is likely mediated by the suppression of ACE gene and protein expression, leading to a lower Ang II level