S-Oxygenation of the thioether organophosphate insecticides phorate and disulfoton by human lung flavin-containing monooxygenase 2
Introduction
Flavin-containing monooxygenase (FMO) is expressed as five gene families in mammals, each with a single member (FMOs 1–5). FMOs in different gene families exhibit 52–58% sequence identity and orthologs across species have 82–97% identity [1], [2], [3]. FMO2 is the major isoform found in lung of most mammals, including non-human primates [4], [5], [6]. This “lung FMO” was originally isolated and characterized from rabbit lung independently by Tynes et al. [7] and Williams et al. [8]. In the lung, FMO2 can account for 10% or more of the total microsomal protein [9]. One of the first observations concerning this lung-specific form of FMO was that it exhibited a substrate specificity demonstrably different from the major FMO in liver [7], [8], [10], [11], [12]. Subsequently, FMO2 has been characterized from mouse, rat, guinea pig, monkey and now human [4], [5], [6], [13], [14], [15], [16], [17]. In humans the majority of individuals possess the FMO2*2 allele, which codes for a truncated (471 amino acids) and non-functional protein (FMO2.2A). Twenty-six percent of individuals of African descent and 5% of Hispanics have at least one FMO2*1 allele, coding for the full-length (535 amino acids), catalytically active protein (FMO2.1) [4], [16], [17], [18]. It has yet to be determined if this genetic polymorphism in expression of FMO2 in human lung is important in the metabolism and toxicity of xenobiotics.
Phorate and disulfoton are thioether-containing organophosphate pesticides that have found wide use in agriculture, primarily on crops such as corn, potatoes, cotton and grains, including wheat. The EPA market estimates for 1999 usage include 2–3 million pounds of the active ingredient of phorate in the US alone; the use of disulfoton is about half that of phorate [19]. Significant exposures to phorate and disulfoton can occur in both occupational settings and to the general public. Occupationally, the primary route of exposure is dermal and inhalation, whereas in the general population it is inhalation, diet and dermal [20], [21]. As inhalation is an important route of exposure, a more thorough understanding of pulmonary pathways for bioactivation and/or detoxication in humans is important.
As with other organophosphates, cytochrome P450 (CYP)-dependent desulfuration to the oxon yields a toxic metabolite with much greater efficacy and potency in inhibition of acetylcholinesterase activity, the major target of organophosphate acute toxicity [22]. CYP can also catalyze formation of the sulfoxide and sulfone metabolites of the parent compound or the oxon [22]. Hodgson and colleagues have published extensive studies on the metabolism of phorate and other pesticides by CYP and FMO [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. In the case of FMO, the sole metabolite is the sulfoxide which is not further oxygenated to the sulfone. FMO also demonstrates a stereoselectivity for formation of the (−) phorate sulfoxide, whereas CYP produces predominately the (+) phorate sulfoxide [27]. Phorate and phorate sulfoxide are both very weak inhibitors of acetylcholinesterase (IC50 of 3100 and 1500 μM, respectively), whereas phorate sulfone is 100 times more potent (40 μM). The oxons (formed only by CYP) are 1000-fold or more potent (IC50 of 3, 0.9 and 0.5 μM for phorate oxon, phorate oxon sulfoxide and phorate oxon sulfone, respectively) [27].
In the case of methiocarb, rat liver FMO (predominantly FMO1) exhibits enantioselectivity toward formation of the (A)-enantiomer which is a more potent acetylcholinesterase inhibitor than the parent compound and the (B)-enantiomer [33]. FMO does not catalyze desulfuration to the oxon. Once the sulfoxide is produced, in the presence of CYP, further metabolism produces the sulfone, the oxon sulfoxide and the oxon sulfone. A high rate of FMO-mediated thioether S-oxygenation, in individuals with the FMO2*1 allele, relative to CYP mediated oxon formation in lung, should decrease toxicity for the majority of thioether-containing organophosphate insecticides.
As for CYP, it has been demonstrated that FMO substrate specificity differs between isoforms [8], [10], [11], [12] and furthermore, that one cannot assume substrate specificity is consistent among orthologs [34]. For this reason, even though phorate and disulfoton have been shown to be substrates for FMOs, including human FMO1 and FMO3 [32] and FMO2 from mouse [13], it is important to characterize the activity of the human FMO2.1 toward these organophosphate insecticides and confirm the identity of the metabolite(s) produced. We confirm that, as with mouse FMO2, human FMO2.1 has high activity toward both of these organophosphates with Km of 32 and 57 μM for disulfoton and phorate, respectively, and the sole metabolites are the S-oxides.
Section snippets
Chemicals
Phorate and disulfoton were obtained from Chem Service Inc. NADPH, EDTA, potassium phosphate, FAD, phenylmethylsulfonyl fluoride (PMSF), glutathione (GSH) and trypan blue were from Sigma. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs. The Bac-to-Bac baculovirus expression system, all components of the BaculoDirect expression system, Sf9 insect cells and media were from Invitrogen.
Expression of FMO2 and preparation of insect cell microsomes
Human FMO2.1 was cloned in pFastBac1 and expressed in Sf9 insect cells with
Results
Incubation of phorate or disulfoton (Fig. 1) with Sf9 insect cell microsomes expressing human FMO2.1 resulted in substrate-dependent NADPH oxidation. Kinetic analysis by double reciprocal plots (data not shown) demonstrated that disulfoton was a better substrate (Km 32 μM, Vmax 71 nmol/min/mg protein), compared to phorate (Km 57 μM, Vmax 63 nmol/min/mg). The metabolism of phorate and disulfoton by expressed rabbit FMO2 assayed under identical conditions yielded somewhat higher Km values of 71 μM for
Discussion
FMOs have been shown to play a significant role in the oxidative metabolism of many pesticides. Among this class of chemicals, thioether-containing organophosphates such as phorate and disulfoton are among the best substrates [13], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [41], [42], [43]. Phorate and disulfoton are S-oxygenated by both CYP and FMO. Oxidative desulfuration to yield the oxon (a more toxic metabolite) is catalyzed by CYP and not FMO. Sulfoxidation
Acknowledgements
This study was supported by PHS grant HL38650. The authors also acknowledge support from the Cell Culture Facility Core and the Mass Spectrometry Facility Core of the Oregon State University Environmental Health Sciences Center (ES 00210). We would also like to acknowledge the laboratory assistance provided by Jonathan Van Dyke.
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