Comparative carboxylesterase activities in infant and adult liver and their in vitro sensitivity to chlorpyrifos oxon

doi:10.1016/j.yrtph.2005.01.004

Regulatory Toxicology and Pharmacology

Volume 42, Issue 1, June 2005, Pages 64-69

https://doi.org/10.1016/j.yrtph.2005.01.004 Get rights and content

Abstract

Maturational expression of carboxylesterase activity in laboratory animals has been correlated with age-related differences in sensitivity to many organophosphorus insecticides including chlorpyrifos. Little information is available, however, on the maturational expression of liver carboxylesterases in humans. Human liver carboxylesterase activity was compared in tissues from infants (2–24 months) and adults (20–36 years). There was no significant difference between mean infant and adult carboxylesterase activities. The carboxylesterase activity rank order was: 2 months < 3 months < 20 years < 24 months < 4 months < 36 years < 21 years < 8 months < 34 years < 35 years. Proteins (3 μg) were separated and blotted using antibodies against rat hydrolase S (HS), human carboxylesterase (HCE) types 1 and 2, and CYP3A4. Again, there were no significant differences in staining density between infant and adult tissues with any isozyme. Aliquots of each sample were pre-incubated (30 min, 37 °C) with chlorpyrifos oxon to evaluate in vitro sensitivity. Based on 95% confidence intervals, no significant differences in IC₅₀ values were obtained in 3-month to 36-year samples (range: 1.42–2.12 nM), while the IC₅₀ was significantly lower in the 2-month sample (0.45 nM). Carboxylesterase activity across samples was correlated with cytochrome b5 content and HS immunosignal but not with other microsomal activities (total cyt P450 content, testosterone hydroxylation, coumarin hydroxylation, and EROD). The results suggest that, in contrast to rodents, human liver carboxylesterase expression changes relatively little during postnatal maturation.

Introduction

Carboxylesterases are important in the detoxification of a number of organophosphorus insecticides (OPs) (Chambers and Chambers, 1990, Chanda et al., 1997, Clement, 1984, Jokanovic, 1989, Maxwell, 1992). Young animals are markedly more sensitive to the acute toxicity of many OPs including chlorpyrifos (Atterberry et al., 1997, Benke and Murphy, 1975, Pope et al., 1991), and the expression of carboxylesterases has been highly correlated with changes in OP sensitivity during both maturation and aging (Benke and Murphy, 1975, Karanth and Pope, 2000, Moser et al., 1998). The inhibition of carboxylesterases can be important in cumulative toxicity with exposure to multiple anticholinesterases including chlorpyrifos (Cohen, 1984, Gupta and Dettbarn, 1993, Karanth et al., 2001). While it has been reported that esterase activities (e.g., butyrylcholinesterase, aryl esterase, and paraoxonase) increase in human plasma during postnatal maturation (Ecobichon and Stephens, 1973), humans have little carboxylesterase activity in the blood. Furthermore, nothing has been reported regarding the maturational expression of human liver carboxylesterases. As noted in the revised cumulative risk assessment for organophosphorus insecticides (EPA, 2002), this gap in information regarding the developmental expression of liver carboxylesterases in humans leads to uncertainty in the estimation of relative risk to some OP insecticides in children.

Human tissue samples for evaluating in vitro xenobiotic metabolism are becoming more readily available. For example, human liver microsomes have been recently used to study the biotransformation of organophosphorus insecticides (Tang et al., 2001, Usmani et al., 2003) and other agents (Akimoto et al., 2004, Morioka et al., 2002). For the present studies, human liver S9 fractions were purchased from a commercial vendor and used to evaluate the age-related expression of carboxylesterases. In vitro effects of chlorpyrifos oxon, the potent oxygen analog of the insecticide chlorpyrifos, on carboxylesterase activity were also evaluated. Relatively minimal differences in carboxylesterase expression or sensitivity to inhibition by chlorpyrifos oxon were noted across age groups, suggesting that this detoxification pathway may have relatively little influence on age-related sensitivity to some organophosphorus insecticides.

Section snippets

Materials and methods

Human liver S9 fractions were obtained from XenoTech LLC, Lenexa, KS. Demographic data as well as information on specific levels of total cytochrome P450, cytochrome b5, 7-ethoxyresorufin O-dealkylation (EROD), coumarin 7-hydroxylation, and testosterone 6β-hydroxylation activities were included with each sample. Chlorpyrifos oxon (>99% purity) was obtained from Chem Service, West Chester, PA. All other chemicals were reagent grade.

Carboxylesterase activity

Carboxylesterase activity was determined by the method of Clement and Erhardt (1990) using p-nitrophenyl acetate or Ecobichon (1970) using α-naphthyl acetate as substrate. Appropriate amounts of S9 fractions were added to buffer (0.1 M Tris–HCl, pH 7.8 at 25 °C containing 2 mM EDTA) and the final volume was adjusted to 1 ml. Samples were pre-incubated at 37 °C for 10 min and the reaction was started by adding 10 μl of 50 mM stock p-nitrophenyl acetate or 125 μl of 10 mM α-naphthyl acetate solution in

Carboxylesterase blotting

Aliquots of S9 fractions (3 μg per well) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes as described previously (Morgan et al., 1994). The blots were initially incubated with non-fat milk (5%) for 60 min and subsequently with antibodies against human carboxylesterase-1 (HCE-1), human carboxylesterase-2 (HCE-2), rat hydrolase S (HS), or CYP3A4. Thereafter, membranes were incubated with alkaline

Statistical analysis

Differences in carboxylesterase activity between infant (2–24 months) and adult (20–36 years) S9 samples were tested for significance by the Mann–Whitney test and/or Student’s t test. Differences in isozyme blotting results between infant and adult samples were also tested by Student’s t test. Significant differences in sensitivity to chlorpyrifos oxon in vitro were estimated by evaluation of 95% confidence intervals about the IC₅₀s. Spearman correlations were determined for comparisons between

Results

Fig. 1 shows the levels of total liver carboxylesterase activity in individual tissues from individuals 2 months to 36 years of age, using p-nitrophenyl acetate as the substrate. Mean carboxylesterase activities in infants (2–24 months of age) and adults (20–36 years of age) were not significantly different between age groups (means ± SEM: infant, 386 ± 87; adult, 641 ± 89, p = 0.074). When evaluated by rank order (Mann–Whitney test), there was also no significant difference between infants and adults (

Discussion

We evaluated the relative expression of human liver carboxylesterase activity and its in vitro sensitivity to the organophosphorus inhibitor chlorpyrifos oxon in tissues from infants (2–24 months) and adults (20–36 years). Relatively little groupwise difference in total esterase activity was noted across ages, and in general, sensitivity to inhibition of esterase activity in vitro by the oxon was not different among the age groups. In tissue from the youngest individual (2 months of age),

Acknowledgments

This work was supported by the Oklahoma State University Board of Regents and Grants R01 ES009119 (C.N.P.) and R01 ES007965 (B.Y.) from the National Institute of Environmental Health Sciences, National Institutes of Health. The contents of the manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.

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Comparative carboxylesterase activities in infant and adult liver and their in vitro sensitivity to chlorpyrifos oxon

Abstract

Introduction

Section snippets

Materials and methods

Carboxylesterase activity

Carboxylesterase blotting

Statistical analysis

Results

Discussion

Acknowledgments

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Fundam. Appl. Toxicol.

Fundam. Appl. Toxicol.

Fundam. Appl. Toxicol.

Chem. Biol. Interact.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Arch. Biochem. Biophys.

Toxicol. Sci.

Toxicology

J. Biol. Chem.