The pyrethroids are one of the most heavily used insecticide classes in the world. It is important to develop sensitive and rapid analytical techniques for environmental monitoring and assessment of human exposure to these compounds. Because major pyrethroids contain a phenoxybenzyl group and phenoxybenzoic acid (PBA) is a common metabolite form or intermediate, PBA might be used as a biomarker of human exposure to pyrethroids. A sensitive and selective immunoassay for the common pyrethroid metabolite PBA was developed. Rabbits were immunized with 3-[4-(3-carboxyphenoxy)phenoxy] N-thyroglobulin ethylamine. All sera were screened against numerous coating antigens. The assay with the least interference and the best sensitivity was optimized and characterized. The average IC50 for free PBA was 1.65 ng/mL. No cross-reactivity was measured to parent pyrethroids and other metabolites. Urine matrix effects can be eliminated by simple dilution. Results from urine samples from exposed workers suggest that this PBA immunoassay might be suitable as a monitoring tool for human exposure to pyrethroids.

Carboxylesterases are enzymes that catalyze the hydrolysis of a wide range of ester-containing endogenous and xenobiotic compounds. Although the use of pyrethroids is increasing, the specific enzymes involved in the hydrolysis of these insecticides have yet to be identified. A pyrethroid-hydrolyzing enzyme was partially purified from mouse liver microsomes using a fluorescent reporter similar in structure to cypermethrin . Compared with their cis-counterparts, trans-permethrin and cypermethrin were hydrolyzed 22-and 4-fold faster, respectively. Of the four fenvalerate isomers the (2R)(␣R)-isomer was hydrolyzed at least 1 order of magnitude faster than any other isomer. However, it is unlikely that this enzyme accounts for the total pyrethroid hydrolysis in the microsomes because both isoelectrofocusing and native PAGE indicate the presence of a second region of cypermethrin-metabolizing enzymes. A second carboxylesterase gene (NCBI accession number NM_133960), isolated during a cDNA mouse liver library screening, was also found to hydrolyze pyrethroids. Both these enzymes could be used as preliminary tools in establishing the relative toxicity of new pyrethroids.

Carboxylesterases hydrolyze a large array of endogenous and exogenous ester-containing compounds, including pyrethroid insecticides. Herein, we report the specific activities and kinetic parameters of human carboxylesterase (hCE)-1 and hCE-2 using authentic pyrethroids and pyrethroid-like, fluorescent surrogates. Both hCE-1 and hCE-2 hydrolyzed type I and II pyrethroids with strong stereoselectivity. For example, the trans-isomers of permethrin and cypermethrin were hydrolyzed much faster than corresponding cis-counterparts by both enzymes. Kinetic values of hCE-1 and hCE-2 were determined using cypermethrin and 11 stereoisomers of the pyrethroid-like, fluorescent surrogates. K m values for the authentic pyrethroids and fluorescent surrogates were in general lower than those for other ester-containing substrates of hCEs. The pyrethroid-like, fluorescent surrogates were hydrolyzed at rates similar to the authentic pyrethroids by both enzymes, suggesting the potential of these compounds as tools for high throughput screening of esterases that hydrolyze pyrethroids. KeywordsCarboxylesterase; Pyrethroid; Fluorescent substrate Synthetic pyrethroid insecticides represented about 17% of the global pesticide market in 2002 [1]. In the coming years, the use of pyrethroids is predicted to further increase with the phase out of the use of organophosphorus insecticides. Additionally, due to the emergence and reemergence of mosquito-vectored diseases such as West Nile fever, Japanese encephalitis, and dengue fever, pyrethroids are being used with increased frequency against adult mosquitoes * Corresponding author. Fax: +1 530 752 1537.E-mail address: bdhammock@ucdavis.edu (B.D. Hammock). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript [2]. Because of the increased agricultural and residential usage of pyrethroids, human exposure to these compounds is also expected to increase.Pyrethroids are ester-containing compounds consisting of various acid and alcohol moieties. Type I pyrethroids are esters of primary alcohols, whereas type II pyrethroids are esters of secondary alcohols that contain a cyano group at the α-carbon of the alcohol moiety. Chiral centers can exist in both the acid and alcohol moieties, leading to the existence of several stereoisomers for each pyrethroid. Ester hydrolysis by carboxylesterases and oxidation by cytochrome P450s are the main detoxification routes of pyrethroids in animals [3]. Differences in the activities of carboxylesterases and P450s between mammals and insects contribute to the low mammalian toxicity of pyrethroids (i.e., pyrethroids are metabolized faster in mammals than in insects) [3]. In general, whole animals or liver microsomes have been used to study pyrethroid metabolism in mammals, and little has been done to identify the specific carboxylesterase isozymes that hydrolyze pyrethroids [4]. Butte and Kemper [5]have shown ester-hydrolytic activities in human serum using pyrethroid-like colorimetric substrates that are not hydrolyzed by acylesterase, ...

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish.