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Arena Pharmaceuticals, Inc., San Diego, California (K.A.U.); and Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina (T.M.C., R.L.R., E.H.)
(Received April 5, 2006; Accepted June 16, 2006)
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Abstract |
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). Human CYP3A4 and CYP1A2 are two of the most important and abundant drug-metabolizing P450 isoforms in human liver microsomes (HLM). On average, CYP3A4 and CYP1A2 account for approximately 40 and 13% of the total P450 in HLM, respectively (Shimada et al., 1994; Lehmann et al., 1998). Human CYP3A4 and CYP1A2 not only metabolize xenobiotics but also are responsible for the metabolism of endogenous compounds, such as steroid hormones. Both human CYP1A2 and CYP3A4 are involved in the metabolism of estradiol (E2) and estrone (Lee et al., 2001, 2003), whereas CYP3A4 plays a predominant role in the metabolism of testosterone (TST), androstenedione, and progesterone (Waxman et al., 1988; Usmani et al., 2003).
In the human female, E2 is the most potent primary circulating estrogen of a group of endogenous estrogen steroids that includes estrone and estriol. During female development, E2 stimulates the growth of female sex organs, regulates and sustains female sexual development and reproductive function, promotes hypertrophy in female breast and male muscle during puberty, initiates the synthesis of specific proteins, and controls fat deposition and distribution in subcutaneous tissues, thereby determining the characteristic female figure. Like TST in males, estrogens are the primary cause of the growth spurt, maturation of long bones, and development of secondary sexual characteristics in females. In the adult, E2 regulates events during the menstrual cycle (growth of endometrial lining), is important during pregnancy and lactation, and contributes to the maintenance of sexual drive and female personality (Constanti et al., 1998; Wilson et al., 1998). Maintaining hormonal balance relies on a number of variables, including the rate of hormone synthesis, interactions among hormones, and rates of secretion, transport, and metabolism by phase I and phase II enzymes. P450s (phase I) are a major element in the maintenance of proper steroid hormone levels in mammalian systems and are the sole subject of this investigation. Exposure to foreign compounds may exert changes in endocrine function both directly (hormone agonists or antagonists) or indirectly (altering circulating levels of hormones by influencing rates of hormone synthesis or metabolism) that can severely affect steroid hormone action (Wilson and LeBlanc, 1998). It follows that perturbation of the P450 system by xenobiotics may in turn affect the subsequent metabolism and disposition of E2. Perturbations in E2 metabolism may affect levels of circulating E2 with possible reproductive and other consequences, including further modulation of the expression of some P450 proteins.
The uses of deployment-related chemicals are essential for the health and well being of deployed forces during both peace-keeping and wartime missions. Human hazards associated with chemicals used to protect personnel during peace and wartime are poorly understood because the definition of cause and effect relationships depends on knowledge of the mechanisms of toxic action and interaction in humans. Following the Gulf War, some veterans reported illnesses that might have been the result of chemical exposures. Some studies of these veterans have concluded that significant correlations between perceived illnesses and chemical use exist (Haley and Kurt, 1997). The reported chemical exposures included the insect repellent N,N-diethyl-m-toluamide (DEET), insecticides such as permethrin and chlorpyrifos used to protect against insect-borne diseases, and the neuroprotective agent pyridostigmine bromide used to protect against possible nerve gas attack. It has been reported that chlorpyrifos and DEET are metabolized by human P450s (Tang et al., 2002; Usmani et al., 2002) and that interactions of deployment-related chemicals can inhibit or induce the P450s involved in their metabolism (Usmani et al., 2002). In a recent study examining the effects of various deployment-related chemicals on TST metabolism, Usmani et al. (2003) reported that organophosphorus pesticides are potent noncompetitive and irreversible inhibitors of TST metabolism caused by CYP3A4 inhibition. Other studies have reported that interaction of Gulf War-related chemicals could produce greater than additive toxicity in rats and mice (Chaney et al., 1997; McCain et al., 1997), increased neurotoxicity in hens associated with increased inhibition of brain acetylcholinesterase and neurotoxicity target esterase (Abou-Donia et al., 1996a,b), and neurobehavioral deficits associated with significant inhibition of brainstem acetylcholinesterase activities in rats (Abou-Donia et al., 2001). Possible detrimental interactions as a result of altered estrogen metabolism have not yet been examined relative to these chemicals, nor have studies been carried out to examine the induction or inhibition by these or related compounds of human P450-mediated metabolism of estrogens, such as E2.
The objective of the present study was to study the inhibition or activation by various deployment-related chemicals of the metabolism of E2 by HLM, CYP1A2, and CYP3A4.
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Materials and Methods |
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Assay Conditions. All the assays were conducted in triplicate, and all the reactions were prewarmed for 5 min at 37°C. All the reactions contained NADPH-regenerating system (0.25 mM NADP, 2.5 mM glucose 6-phosphate, and 2 U/ml glucose-6-phosphate dehydrogenase) and 100 mM potassium phosphate buffer (pH 7.4) containing 5 mM ascorbic acid and 3.3 mM MgCl2. Final concentrations of HLM were 1 mg/ml, and final concentrations of CYP3A4 and CYP1A2 were 50 pmol/ml. The final reaction volume was 250 µl, and all the reactions were terminated after 20 min by the addition of 150 µl of methanol and vortexing. After 5-min centrifugation at 21,000g in a microcentrifuge, the supernatants were analyzed for E2 metabolite concentrations by HPLC. The protein concentrations and incubation times used in the assays were found to be in the linear range in preliminary experiments. No metabolites were detected when incubations were carried out in the absence of an NADPH-generating system.
In Vitro E2 Metabolism. Enzyme kinetic assays using HLM or the recombinant isoforms CYP1A2 or CYP3A4 were performed by incubation of serial concentrations of E2 (final concentrations 3.125–200 µM) with an NADPH-generating system in potassium phosphate buffer and incubated at 37°C for 5 min. The reactions were initiated by the addition of ice-cold HLM (final protein concentration 1 mg/ml), CYP1A2, or CYP3A4. No measurable lag phase was observed.
To determine the metabolic activities of individual HLM, incubations of E2 (final concentration 50 µM) were conducted for 20 min at 37°C using 1 mg protein/ml. To differentiate between the relative concentrations of CYP1A2 and CYP3A4 and the metabolism of E2 in individual HLM, a specific CYP3A4 inhibitor, ketoconazole (final concentration 2 µM), was added to the reaction mixture simultaneously with 50 µM E2.
Preliminary Inhibition Studies. The effects of test chemicals (chlorpyrifos, fonofos, phorate, DEET, fipronil, imidacloprid, deltamethrin, permethrin, carbofuran, carbaryl, naphthalene, and pyridostigmine bromide) on E2 metabolism were examined using HLM, CYP1A2, or CYP3A4 after preincubation with the test compounds in otherwise complete mixtures lacking only the substrate E2. HLM, CYP3A4, or CYP1A2 was incubated with individual test compounds (final concentration 50 µM) for 5 min at 37°C before adding E2 (final concentration 50 µM). All the inhibitors, except pyridostigmine bromide, were added in 2.5 µl of acetonitrile with an equal volume of acetonitrile being added to the control incubations. Pyridostigmine bromide was added in 2.5 µl of water. Because of the likelihood that phosphorothioates are metabolized to irreversible P450 inhibitors, preincubation is tested as a variable with these chemicals. Preliminary experiments indicated little or no difference between preincubation and coincubation for other potential inhibitors. Only those chemicals that were capable of greater than 50% inhibition of E2 metabolism at a concentration of 50 µM were studied further.
Inhibition Kinetics of Deployment-Related Chemicals on E2 Metabolism by CYP3A4 and CYP1A2. The chemicals for which IC50 values were below 25 µM as determined by preliminary incubation studies were analyzed further for inhibitory potential. Range-finding assays were conducted for chlorpyrifos, fonofos, phorate, deltamethrin, and permethrin (0.39–200 µM) by incubating with CYP3A4 for 5 min at 37°C before adding E2 (final concentration 50 µM). The kinetic study of E2 using varying E2 concentrations (6.25–100 µM) was performed using CYP3A4, which was preincubated with chlorpyrifos or fonofos (0.78, 1.56, or 3.125 µM) for 5 min at 37°C.
For CYP1A2, range-finding assays were conducted for chlorpyrifos and fonofos as described above. Concentrations of chlorpyrifos (0.5 and 2.0 µM) and fonofos (12.5 and 50.0 µM) were preincubated with CYP1A2 for 5 min at 37°C before adding E2 (final concentration 3.125–100 µM) to measure their inhibition of CYP1A2 metabolism of E2. In all the inhibition studies involving preincubation with the inhibitor, the NADPH-generating system was present during the preincubation.
Preincubation Time- and Concentration-Dependent Inactivation by Chlorpyrifos of CYP3A4 Metabolism of E2. These studies were conducted using the same conditions as described in the assay conditions (50 µM E2; 20-min incubation at 37°C) but varying concentrations (0, 1, 2, 5, 10, and 50 µM CPS) and preincubation times (0, 1, 2, 3, 4, and 5 min). The generation of 2-OHE2 (the major metabolite of E2) was compared in this assay and conducted in duplicate. Inactivation parameters (kinact and KI) were estimated as described in previous studies (Silverman, 1995; Heydari et al., 2004). The rate constant for initial inactivation (kobs) at each concentration of inhibitor (CPS) was calculated from the slopes in the regression plots (–kobs) (natural logarithm of remaining activity as percentage of control activity versus preincubation time). The inactivation values (kinact and KI) were calculated as described in Heydari et al. (2004); kobs = kinactx [I]/(KI + [I]), where [I] is the inhibitor concentration, kinact is the maximum rate constant for inactivation, and KI is the inhibitor concentration that produces half-maximal rate of inactivation.
Inhibition Kinetics of Carbaryl and Its Metabolites on E2 Metabolism by CYP1A2. Before the Ki (inhibition constant) determination was done, range-finding assays were conducted for the inhibition by carbaryl and its metabolites of the metabolism of E2 by CYP1A2. Varying concentrations (1.56–50 µM) of carbaryl, 4-hydroxycarbaryl, 5-hydroxycarbaryl, and carbaryl methylol were preincubated or coincubated with CYP1A2. Preincubation experiments with inhibitors were conducted similarly to those described above. For coincubation experiments, carbaryl or its metabolites were incubated with E2 (final concentration 50 µM). The reactions were initiated by the addition of ice-cold CYP1A2. For Michaelis-Menten plots, carbaryl (6.25 and 12.5 µM), 4-hydroxycarbaryl (6.25 and 12.5 µM), and carbaryl methylol (12.5 and 25 µM) were coincubated with E2 (final concentration 3.125–100 µM). The reactions were initiated by the addition of ice-cold CYP1A2.
Inhibition Kinetics of Naphthalene and Its Metabolites on E2 Metabolism by CYP1A2. Before the Ki determination was done, range-finding assays were conducted for the inhibition by naphthalene and its metabolites of the metabolism of E2 by CYP1A2. Varying concentrations of naphthalene (3.125–25 µM), 1-naphthol (0.78–25 µM), 2-naphthol (25 µM), trans-1,2-dihydro-1,2-naphthalenediol (25 µM), 1,2-napthoquinone (25 µM), 1,4-naphthoquinone (25 µM), and 1,4-dihydroxynapthlene (25 µM) were preincubated or coincubated with CYP1A2. The preincubation and coincubation experiments with naphthalene and its metabolites were conducted similarly as described above. For Michaelis-Menten plots, naphthalene (1.56, 3.125, and 6.25 µM) was preincubated with CYP1A2 for 5 min at 37°C before adding E2 (final concentration 3.125–100 µM).
For Michaelis-Menten plots, 1-naphthol (1.56 and 3.125 µM) was coincubated with E2 (final concentration 3.125–100 µM). The reactions were initiated by the addition of ice-cold CYP1A2.
To show whether 1-naphthol inhibition is reversible or irreversible, incubations with and without 1-naphthol (3.125 µM) were conducted with E2 (final concentration 50 µM). The reactions were initiated by the addition of varying concentrations of ice-cold CYP1A2 (3.125–12.5 pmol).
Analysis of 2-OHE2 and 6ß-Hydroxytestosterone by HPLC. Analysis of the E2 metabolite 2-OHE2 was performed with an HPLC system coupled with in-line UV detection as described previously (Suchar et al., 1995). The HPLC system consisted of a Waters 2690 separation module and a Waters UV photodiode array detector (model 2996). All the system components were controlled through the Waters Powerline firmware. Data were collected via a Waters system controller and analyzed using Waters Empower software. The solvent system for separation of E2 and 2-OHE2 consisted of acetonitrile (solvent A), 0.1% acetic acid in water (solvent B), and 0.1% acetic acid in methanol (solvent C). The solvent gradient (solvent A/solvent B/solvent C) used for eluting E2 and 2-OHE2 was as follows: 8 min of isocratic at 16:68:16, 7 min of a concave gradient (curve number 9) to 18:64:18, 13 min of a concave gradient (curve number 8) to 20:59:21, 10 min of a convex gradient (curve number 2) to 22:57:21, 13 min of a concave gradient (curve number 8) to 58:21:21, followed by a 0.1-min step to 92:5:3 and a 3.9-min isocratic period at 92:5:3. The gradient was returned to the initial condition (16:68:16) for 2 min and held for 3 min before analyzing the next sample. The flow rate was 1.2 ml/min. E2 and 2-OHE2 were separated by an Ultracarb 5 ODS (octadecylsilane) column (150 x 4.6 mm, Phenomenex, Rancho Palos Verdes, CA) at 30°C and detected at 280 nm. The limit of detection for 2-OHE2 was approximately 0.04 µM. Concentrations of 2-OHE2 were obtained from the chromatographic peak area from a standard curve (0.15–20.0 µM).
TST and its metabolite, 6ß-hydroxytestosterone, were separated using the method described by Usmani et al. (2003). E2 and its metabolites eluted after 6ß-hydroxytestosterone and TST.
Data Analysis and Statistics. The apparent Km and Vmax parameters were calculated using the SigmaPlot Enzyme Kinetics Module for Windows, version 1.1 (SPSS Inc., Chicago, IL). The Ki values were estimated by nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SigmaPlot Enzyme Kinetics Module for Windows, version 1.1 (SPSS Inc.). The mode of inhibition was established by comparing the statistical results including the r2 information criterion values of different inhibition models and by selecting the one with the best fit. Furthermore, the mode of inhibition was established by comparing the 95% confidence intervals that did not overlap for Km and Vmax values. Significant differences between data sets were determined by Student's t test using SigmaPlot for Windows, version 8.0 (SPSS Inc.).
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Results |
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E2 Metabolism in Single-Donor HLM. Incubations of E2 with individual donor HLM (6 male and 11 female) showed as much as 36-fold variability in metabolism among individuals (Fig. 2). Phenotype data based on metabolic activities of these individuals was used to derive correlations between P450 isoform content and E2 metabolic activity. CYP3A4 was the isoform with the best correlation (r2 = 0.87). All the other correlations between E2 hydroxylation activity and specific P450 isoforms were less than 0.40. To determine the CYP1A2 contribution to E2 metabolism among individual HLM, ketoconazole (2 µM), a specific CYP3A4 inhibitor, was used to inhibit CYP3A4 activity. Preliminary observations with ketoconazole indicated that 2 µM inhibited less than 5% of CYP1A2 activity (data not shown). In the presence of ketoconazole, variation among individuals was reduced to 12-fold, and the correlation of CYP1A2 with E2 metabolism increased to 0.81 (Fig. 2).
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In a similar manner, inhibition of CYP1A2 metabolism of E2 by chlorpyrifos and fonofos involved a 5-min preincubation before the addition of varying concentrations of E2. As shown for CYP3A4 (Fig. 5), Michaelis-Menten plots for CYP1A2 also indicated noncompetitive inhibition (Fig. 7).
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In the determination of IC50 values, varying concentrations of naphthalene and its metabolites were preincubated and coincubated with CYP1A2. Preincubation of naphthalene increased the inhibition of E2 metabolism by nearly 2-fold compared with the coincubation of naphthalene with E2, indicating that a reactive metabolite is involved (data not shown). No differences were observed in the IC50 values when 1-naphthol was preincubated or coincubated with E2 (data not shown). 2-Naphthol, 1,2-napthoquinone, 1,4-naphthoquinone, and 1,4-dihydronaphthalene (25 µM) inhibited E2 (50 µM) metabolism ca. 85 to 100%, whereas trans-1,2-dihydro-1,2-naphthalenediol had no inhibitory effect on E2 metabolism (data not shown). To determine the Ki value and to investigate the type of inhibition of CYP1A2 by naphthalene on E2 metabolism, naphthalene was preincubated for 5 min with CYP1A2 before adding varying concentrations of E2. The Michaelis-Menten plot showed that the Vmax values were significantly reduced without affecting Km values, indicative of a noncompetitive inhibition of E2 metabolism by naphthalene (Fig. 10). To investigate the type of inhibition of CYP1A2 by 1-naphthol on E2 metabolism, 1-naphthol was coincubated with CYP1A2 and varying concentrations of E2. 1-Naphthol, a predominant naphthalene metabolite, was shown to be a noncompetitive inhibitor of E2 metabolism (Fig. 11). Further investigation of noncompetitive reversible or nonreversible inhibition data revealed that the inhibition of 2-OHE2 by 1-naphthol is nonreversible (data not shown).
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Discussion |
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; Lee et al., 2001, 2003). The phenotyped HLM data and the mean metabolic intrinsic clearance rates, as estimated by Vmax/Km, corroborate earlier findings (Yamazaki et al., 1998; Lee et al., 2001, 2003) that both CYP3A4 and CYP1A2 play major roles in the metabolism of E2 in human liver, and the content of these two P450s in HLM determine which isoform is more important. However, CYP3A4 may play a more important role than CYP1A2 because on average CYP3A4 accounts for approximately 40% of the total P450 in HLM. Our kinetic data presented in this study for CYP1A2 and CYP3A4 are in general agreement with the previous reports showing that CYP1A2 is more active than CYP3A4 in E2 metabolism (Yamazaki et al., 1998; Badawi et al., 2001).
CYP3A4 is one of the most important and abundant P450 isoforms in human liver and has broad substrate specificity. CYP3A4 not only metabolizes xenobiotics but also is responsible for the metabolism of endogenous compounds such as steroid hormones, including TST and E2 (Lee et al., 2003; Usmani et al., 2003). Because TST and E2 are predominantly metabolized by CYP3A4, both can potentially compete for the same catalytic site of CYP3A4. The usual interaction between two different substrates for the same enzyme is competitive inhibition. But because the Km values for TST and E2 are different (Km = 108 and 24 µM for TST and E2, respectively), interactions between these substrates may be complicated because of the allosteric characteristics of CYP3A4 (Shimada and Guengerich 1989; Lee et al., 1995; Usmani et al., 2003; Williams et al., 2004). The recent demonstration of a relatively large substrate binding catalytic site by crystal structure elucidation is consistent with the capacity of CYP3A4 to accommodate large molecules and possibly more than one substrate (Yano et al., 2004).
Endogenous steroids, such as E2, always exist in vivo, and considerable amounts of these steroids are metabolized by the P450s expressed in the human liver, where foreign compounds are mainly metabolized. Because TST and E2 both have important, and different, effects in vivo, the inhibition of the metabolism of both of these steroid hormones must be considered in detail. If xenobiotics substantially alter enzymes such as CYP3A4 and CYP1A2, they may affect the rate of E2 metabolism, ultimately disrupting E2 homeostasis. Preincubation of pooled HLM, CYP3A4, and CYP1A2 with organophosphorus compounds, such as chlorpyrifos and fonofos, resulted in the extensive inhibition of the production of 2-OHE2, a major E2 metabolite. Chlorpyrifos and fonofos inhibited 2-OHE2 formation noncompetitively and are among the most potent inhibitors of the P450-dependent oxidation of E2 yet described. Organophosphorus pesticides, such as chlorpyrifos and fonofos, are activated by a P450-catalyzed desulfuration reaction (Fukuto, 1990). The sulfur atom released from these pesticides in this reaction is highly reactive and is believed to bind immediately to the heme iron of P450 and inhibit its activity (Norman et al., 1974, Halpert et al., 1980, Neal, 1980; Neal and Halpert, 1982; Butler and Murray, 1997). Our previous study showed that organophosphorus pesticides are potent noncompetitive and irreversible inhibitors of TST metabolism by HLM and CYP3A4 (Usmani et al., 2003).
Enzyme activity is often modulated by inhibition or induction, either condition modifying the extent to which xenobiotics or endogenous substrates are metabolized (Guengerich, 1997; Szklarz and Halpert, 1998). Inhibition may, in some interactions, be more serious than enzyme induction because inhibition occurs more rapidly than induction (Guengerich, 1997). Preincubation of CYP3A4 with chlorpyrifos and fonofos resulted in almost complete inhibition of E2 metabolism to 2-OHE2. Preincubation of CYP1A2 with chlorpyrifos resulted in almost complete inhibition of 2-OHE2 formation, whereas fonofos was not as potent an inhibitor. The kinetics of inactivation of E2 metabolism by CPS confirms that CPS is a mechanism-based inactivator of CYP3A4 in these studies. Preincubation of CPS in the presence of an NADPH-generating system increased the inhibitory effect in a time- and concentration-dependent manner. The significant reduction of CYP3A4 activity toward E2 metabolism by CPS at time 0' suggests that the binding affinity of the inhibitor (CPS) is higher than that of the substrate (E2) with the CYP3A4 isoform and that the initial rate of inhibition is rapid. The relatively low KI value (high inhibitory potency) of CPS toward CYP3A4 metabolism of E2 appears to confirm the strong binding affinity of CPS with this isoform. Therefore, the possibility exists that inhibition of CYP3A4 and CYP1A2 by these chemicals could lead to higher levels of E2 and alter hormonal properties. The in vivo importance of these observations will require further studies.
Carbaryl is a widely used anticholinesterase carbamate insecticide. In an in vitro study, 5-hydroxycarbaryl, 4-hydroxycarbaryl, and carbaryl methylol were identified as the major metabolites of carbaryl produced by HLM (Tang et al., 2002). P450 isoform data indicated that CYP1A1, CYP1A2, CYP2B6, CYP2C19, and CYP3A4 are the most active isoforms in human metabolism of carbaryl (Tang et al., 2002). In our preliminary inhibition studies, we found that preincubation of pooled HLM and CYP1A2 with carbaryl significantly inhibited E2 metabolism. Because carbaryl is metabolized by CYP1A2 while also showing significant inhibition of E2 metabolism, we expected competitive interactions between E2 and carbaryl or its metabolites. However, carbaryl and its metabolites inhibited formation of 2-OHE2 noncompetitively in our coincubation studies. The Ki values indicated that carbaryl and 4-hydroxycarbaryl were much better inhibitors of 2-OHE2 than carbaryl methylol. Although there is no obvious explanation of the slight activation of E2 at low concentrations of carbaryl, 4-hydroxycarbaryl, and carbaryl methylol, it may be noted that this is a common phenomenon in the oxidation of TST by HLM (Usmani et al., 2003).
The polycyclic aromatic hydrocarbon naphthalene is an industrial chemical, an environmental pollutant, and a component of jet fuel and has considerable toxicological importance because of widespread human exposure and its potential to form toxic and carcinogenic metabolites in humans (Wilson et al., 1996). We recently showed that CYP1A2 is the most efficient of the 15 different isoforms tested for their ability to metabolize naphthalene to its two major metabolites in HLM, 1-naphthol and 1,2-dihydro-1,2-naphthalenediol (Cho et al., 2006). In this study, preincubations of naphthalene with pooled HLM and CYP1A2 significantly inhibited E2 metabolism. Because naphthalene is metabolized by CYP1A2 and also significantly inhibited E2 metabolism, we expected a competitive interaction between E2 and naphthalene or its metabolite. However, this study showed noncompetitive inhibition of E2 metabolism. Coincubation of 1-naphthol, a well known cytotoxic metabolite of naphthalene, resulted in noncompetitive and irreversible inhibition of E2 metabolism.
In conclusion, the hydroxylation of E2 by CYP3A4 and CYP1A2 isoforms indicates important functions for these enzymes other than detoxification of xenobiotics. The deployment-related and other chemicals used in this study, including pesticides, caused a marked modification of P450-mediated E2 metabolism in vitro. Organophosphorus pesticides were very potent inhibitors of the production of 2-OHE2 and inhibited E2 metabolism noncompetitively. Carbaryl and some of its metabolites inhibited E2 metabolism noncompetitively. Naphthalene inhibited E2 metabolism noncompetitively, an effect probably caused by 1-naphthol, a naphthalene metabolite. 1-Naphthol inhibits E2 metabolism noncompetitively and irreversibly. It should be noted that in vivo toxicokinetic data are not available for these chemicals in humans, and thus the effects noted indicate only the potential for in vivo effects.
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This work was supported by U.S. Army Cooperative Agreement DAMD 17-00-2-0008. Parts of the studies were presented at the 7th International ISSX Meeting in Vancouver, Canada, 2004 and the 44th Annual Meeting of the Society of Toxicology in New Orleans, 2005.
ABBREVIATIONS: P450, cytochrome P450; HLM, human liver microsome(s); E2, estradiol; TST, testosterone; DEET, N,N-diethyl-m-toluamide; 2-OHE2, 2-hydroxyestradiol; HPLC, high-performance liquid chromatography; CPS, chlorpyrifos.
1 Deceased. Dr. Rose died in a tragic accident on May 23, 2006. 
Address correspondence to: Ernest Hodgson, Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, NC 27695. E-mail: ernest_hodgson{at}ncsu.edu
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, statistically significant different when compared with control (P < 0.05).
