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