In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol

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Abstract

Carbofuran is a carbamate pesticide used in agricultural practice throughout the world. Its effect as a pesticide is due to its ability to inhibit acetylcholinesterase activity. Though carbofuran has a long history of use, there is little information available with respect to its metabolic fate and disposition in mammals. The present study was designed to investigate the comparative in vitro metabolism of carbofuran from human, rat, and mouse liver microsomes (HLM, RLM, MLM, respectively), and characterize the specific enzymes involved in such metabolism, with particular reference to human metabolism. Carbofuran is metabolized by cytochrome P450 (CYP) leading to the production of one major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in the oxidation to 3-hydroxycarbofuran is significantly less in HLM (Km = 1.950 mM) than in RLM (Km = 0.210 mM), or MLM (Km = 0.550 mM). Intrinsic clearance rate calculations indicate that HLM are 14-fold less efficient in the metabolism of carbofuran to 3-hydroxycarbofuran than RLM or MLM. A screen of 15 major human CYP isoforms for metabolic ability with respect to carbofuran metabolism demonstrated that CYP3A4 is the major isoform responsible for carbofuran oxidation in humans. CYP1A2 and 2C19 are much less active while other human CYP isoforms have minimal or no activity toward carbofuran. In contrast with the human isoforms, members of the CYP2C family in rats are likely to have a primary role in carbofuran metabolism. Normalization of HLM data with the average levels of each CYP in native HLM, indicates that carbofuran metabolism is primarily mediated by CYP3A4 (percent total normalized rate (% TNR) = 77.5), although CYP1A2 and 2C19 play ancillary roles (% TNR = 9.0 and 6.0, respectively). This is substantiated by the fact that ketoconazole, a specific inhibitor of CYP3A4, is an excellent inhibitor of 3-hydroxycarbofuran formation in HLM (IC50: 0.31 μM). Chlorpyrifos, an irreversible non-competitive inhibitor of CYP3A4, inhibits the formation of 3-hydroxycarbofuran in HLM (IC50: 39 μM). The use of phenotyped HLM demonstrated that individuals with high levels of CYP3A4 have the greatest potential to metabolize carbofuran to its major metabolite. The variation in carbofuran metabolism among 17 single-donor HLM samples is over 5-fold and the best correlation between CYP isoform activity and carbofuran metabolism was observed with CYP3A4 (r2 = 0.96). The interaction of carbofuran and the endogenous CYP3A4 substrates, testosterone and estradiol, were also investigated. Testosterone metabolism was activated by carbofuran in HLM and CYP3A4, however, less activation was observed for carbofuran metabolism by testosterone in HLM and CYP3A4. No interactions between carbofuran and estradiol metabolism were observed.

Introduction

Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) is the most commonly used carbamate in agriculture and forestry. Carbofuran is a broad spectrum pesticide that kills insects, mites, and nematodes on contact or after ingestion. The mechanism of toxicity is anticholinesterase activity [1], resulting in accumulation of acetylcholine in synapses and subsequent malfunction of the nervous system. In addition to neurotoxic effects, carbofuran has been demonstrated to influence steroid metabolism in mammals [2], [3], [4]. Recent epidemiological studies indicate that individuals with high levels of exposure to carbofuran and other carbamate pesticides may have increased risk for lung cancer (C.R. Alavanja, personal communication) and non-Hodgkins lymphoma [5].

The most recent extensive review of carbofuran toxicity and metabolism is that by Gupta [6]. Primary mammalian metabolites include 3-hydroxycarbofuran, 3-ketocarbofuran, 3-ketocarbofuran-7-phenol, and carbofuran phenol. Each of these metabolites is found in the free state and also as sulfate and glucuronide conjugates which are excreted in the urine. In vivo studies involving oral exposure of rats to carbofuran demonstrated that the predominant metabolite in the bile was 3-hydroxycarbofuran glucuronide, a metabolite which may be cleaved to yield a potent anticholinesterase aglycone. Since the enterohepatic cycling of glucuronides involves cleavage of the conjugate in the gut, biliary excretion may actually lead to increased systemic activity of toxic carbofuran metabolites since both carbofuran and 3-hydroxycarbofuran are equally potent cholinesterase inhibitors [6].

Metabolism plays an important role in the determination of pesticide toxicity. Hepatic metabolism of carbofuran in humans has neither been previously investigated in vitro nor have the contributions of CYP isoforms to metabolic pathways been elucidated. An understanding of the metabolic pathways and the varying contributions of specific CYP isoforms involved will enable better understanding of differences in metabolism among individuals as well as among subpopulations and will provide important information relative to metabolic interactions of carbofuran with other chemicals.

Studies of pesticide metabolism in humans can provide important information on differences between humans and laboratory animals in metabolism. Rodent studies have been useful for decades for predicting human health hazards associated with pesticide use. However, studies conducted in experimental animals can sometimes be misleading since human xenobiotic-metabolizing enzymes often differ dramatically from those of experimental animals, rendering such extrapolations of little value. The present in vitro study was designed to: (1) compare the metabolism of carbofuran in human, rat, and mouse liver microsomes; (2) elucidate human CYP isoforms responsible for metabolism of carbofuran; (3) determine potential differences in oxidation activities among individual human liver microsomes; (4) examine potential interactions of carbofuran with endogenous chemicals that are substrates for the same enzymes.

Section snippets

Chemicals

Carbofuran was purchased from Chem Service Inc. (West Chester, PA). Carbofuran metabolites, 3-hydroxycarbofuran, 3-ketocarbofuran, 3-ketocarbofuran phenol, 3-hydroxycarbofuran phenol, and carbofuran phenol were a gift from FMC (Princeton, NJ). Testosterone, 6β-hydroxytestosterone, 17β-estradiol, 2-hydroxyestradiol were purchased from Steraloids (Newport, RI). HPLC grade acetonitrile and water were purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals, if not specified, were

Enzyme kinetics of carbofuran metabolism in liver microsomes and human hepatocytes

In vitro incubations of carbofuran with HLM, RLM, and MLM resulted in the production of one major and two minor metabolites (Fig. 1). The major metabolite of carbofuran incubations in microsomal samples was 3-hydroxycarbofuran, while the minor metabolites included 3-keto-7-phenol and one unidentified minor metabolite. Further investigations involving incubations of 3-hydroxycarbofuran with HLM demonstrated that it is metabolized by CYPs to a minor metabolite, 3-ketocarbofuran, which is

Discussion

The predominant metabolite of carbofuran in HLM, RLM, MLM, and human hepatocytes is 3-hydroxycarbofuran. The enzyme kinetic studies indicated that Km values for carbofuran metabolism in HLM and human hepatocytes are ca. 9.5-fold and 3.6-fold lower than RLM and MLM, respectively. The mean metabolic intrinsic clearance rates, as estimated by Vmax/Km, indicate that RLM and MLM metabolize carbofuran ca. 15-fold more efficiently than HLM, indicating that, in general, humans are not as active as

Acknowledgments

This project was supported in part by NIOSH grant OH07551-ECU.

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