CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Received March 8, 2004; accepted May 6, 2004
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ABSTRACT |
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Key Words: ethylene; ethylene oxide; cytochrome P450; metabolism; CYP 2E1.
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INTRODUCTION |
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The pharmacokinetic behavior of ethylene and its conversion to EO has been a major consideration in risk assessment of ethylene. The uptake of ethylene on inhalation exposure is limited, and the conversion of ethylene to EO has been reported to be saturated at approximately 1000 ppm in the rat (Bolt and Filser, 1984; Bolt et al., 1984
).
There is considerable evidence that ethylene is metabolized to EO by cytochrome P450, primarily in the liver. However, there is potential for ethylene to undergo metabolism in other tissues. Cytochrome P450 2E1 is probably the major isozyme responsible for the oxidation of ethylene, although others may contribute. Diethyldithiocarbamate, a selective inhibitor of CYP 2E1 (Guengerich et al., 1991), administered to rats completely inhibited metabolic elimination of ethylene (Bolt et al., 1984
). The involvement of other isozymes is possible since metabolic elimination in rats in vivo could be induced by Aroclor 1254 pretreatment (Filser and Bolt, 1983
) and inhibited in vitro in rat liver microsomes by ß-naphthoflavone (Schmiedel et al., 1983
).
Investigation of the relationship between exposure to ethylene and production of EO has revealed numerous unusual findings. During an exposure of male Sprague-Dawley rats to a high concentration of ethylene in a closed chamber, the concentration of EO in the chamber air peaked at about 1 h and then declined by 2.5 h, reaching a steady state at approximately half of the peak concentration (Filser and Bolt, 1984). During a 6-h exposure of male F344 rats to 600 ppm ethylene, a peak blood concentration of 3 µg/g was seen for EO at 8 min (Maples and Dahl, 1993
). This dropped to 0.6 µg/g 4 min later and then remained constant. Cytochrome P450 content, measured by the reduced CO difference spectrum, dropped to 94% of control values by 20 min and to 68% by 360 min. From this study, it would appear that there is a loss in metabolic activity preceding a loss of spectrally detected cytochrome P450. Limitations of this study are that no enzyme activity measurements were conducted and spectral P450 measurements were performed on a single pooled sample for each group. The time frame for the decrease in metabolism reflected in blood concentrations was considerably sooner than that observed with exhaled EO. Incubation of microsomes in vitro with ethylene caused the inhibition of cytochrome P450 and degradation of the heme prosthetic group of cytochrome (Ortiz de Montellano and Mico, 1980
; Ortiz de Montellano et al., 1981
). The resulting product was identified as N-(2-hydroxyethyl)protoporphyrin IX.
Our aim was to investigate the hypothesis that CYP 2E1 is inhibited by ethylene exposure. We addressed the issues of whether the observations of Maples and Dahl (1993) could be reproduced, whether the effects could be observed over a range of exposure concentrations, and what were the dose-response and temporal characteristics of the effect.
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MATERIALS AND METHODS |
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Animals. This study was conducted under federal guidelines for the use and care of laboratory animals (National Research Council, 1996) and was approved by the Institutional Animal Care and Use Committee of the CIIT Centers for Health Research (CIIT). Animals were housed in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. For collection of blood during exposure (for measurement of EO), rats with implanted jugular vein cannulas were used. Cannulated male CDF (F344)/CrlBR rats (F344) were obtained from Charles River Laboratories (Raleigh, NC). Jugular vein cannulas were implanted at the vendor and the rats were shipped to CIIT the day after surgery. The animals were approximately 10 weeks old at the time of exposure. Noncannulated male F344 rats were also purchased from Charles River Laboratories for the collection of liver samples. All animals were housed in filter-capped cages containing Alpha-Dri contact bedding (Shepherd Specialty Papers, Inc., Kalamazoo, MI). Rats were housed singly and bedding was changed twice per week. The room temperature was maintained at approximately 72 ± 7°F, and relative humidity was maintained at approximately 3070%. Automatic light controls provided fluorescent lighting for a photoperiod of approximately 12 h. Animals were fed pelleted standard NIH-07 rodent chow (Zeigler Bros., Gardners, PA) ad libitum, except during inhalation exposure. Water was filtered through a Hydro Picosystem filter system and was provided ad libitum from water bottles, except during inhalation exposure.
Inhalation exposures. Ethylene exposure atmospheres (300, 600, and 1000 ppm) were generated by mixing ethylene (13% in nitrogen) from a certified gas cylinder (Scott Specialty Gases) with compressed air. The mixture of ethylene in air passed into a stainless steel, flow-past, nose-only unit (NOU) located inside a down draft hood (Cannon et al., 1983). Air flows through the NOU were adjusted to provide a minimum flow of approximately 0.5 l/min at each open exposure port. Rats were exposed in open-style, nose-only tubes. Ethylene concentrations were monitored with a calibrated infrared spectrophotometer (MIRAN 1A, The Foxboro Co., Foxboro, MA).
For the determination of blood EO concentrations, groups of 15 male rats with jugular vein cannulas were exposed to 300, 600, or 1000 ppm ethylene. Approximately 0.2 ml blood samples were drawn at 3, 5, 7.5, 10, 15, and 30 min and at 1, 2, 4, and 6 h during the exposure. The blood samples were immediately placed in preweighed, 12-ml, crimp-seal vials and sealed. The samples were weighed and stored on ice until analysis. Blood collected prior to the exposure served as the control.
For the determination of cytochrome P450 and associated activities, two studies were performed. Initially, exposure at 1000 ppm ethylene was conducted on 44 noncannulated F344 rats. Groups of four rats were removed from the exposure tower and euthanized by exposure to CO2 at 5, 10, 15, and 30 min and at 1, 2, 4, and 6 h after the start of exposure. Additional groups of four rats were exposed to 1000 ppm ethylene for 6 h and euthanized with CO2 at 12, 24, and 48 h after the start of inhalation exposure. A control group (16 rats) was exposed to air for 6 h in the nose-only exposure system and euthanized at 6, 12, 24, and 48 h (four rats at each time point) after the start of exposure. Thus, to minimize the number of animals used, a single control group exposed to air for 6 h was used for comparison with all of the rats exposed to ethylene for up to 6 h. However, the 12-, 24-, and 48-h time points each had time-matched controls. A second study was performed in a manner similar to the first with exposure to 0, 300, and 600 ppm ethylene. Groups of 20 noncannulated F344 rats were exposed for up to 6 h to ethylene at each concentration, and four rats were euthanized at 2, 4, 6, 12, and 24 h after the start of exposure. Livers were placed in ice-cold 1.15% KCl until microsome preparation.
Analysis of blood EO. EO in blood was quantitated by gas chromatography (GC) with flame ionization detection (FID) in a manner like that described previously (Brown et al., 1996). Analysis was conducted on a Hewlett Packard 5890 GC with an HP-7694 headspace autosampler and an HP 1100 Chemstation data system (Hewlett Packard, Palo Alto, CA). Chromatography was conducted on a 30 m x 0.53 mm, 1-µm film thickness DB-Wax column: (Agilent Technologies, Palo Alto, CA). Helium was used as carrier gas at a flow rate of 5 ml/min. Samples (1.0 ml) were analyzed with an injector temperature of 150°C, a detector temperature of 300°C, and an initial oven temperature of 35°C. Samples were removed from ice and incubated for 10 min in the headspace autosampler oven at a temperature of 37°C. EO eluted at approximately 2.6 min. After 3.5 min, a temperature gradient of 70°C/min to 100°C was used to recondition the column for the next injection. Standard curve development was conducted with gas bags with defined concentrations of EO. Blood samples spiked with EO were used to develop appropriate analytical conditions and a standard curve. Standards were prepared with vials containing blood to which defined volumes of EO:air mixtures were added. No correction was applied for the potential loss of EO during incubation in the headspace autosampler, since this loss was estimated to be approximately 7% based on the first order rate constant for loss of EO in rat blood of 100 min (Brown et al., 1996
).
Liver microsome isolation. Livers were removed and weighed and placed on ice in 3 volumes of ice-cold 1.15% KCl. Livers were scissors-minced and homogenized with a Potter-Elvehjem (Kimble-Kontes, Vineland, NJ) homogenizer (five strokes). The homogenate was centrifuged for 20 min at 20,000 x g (13,000 rpm) at 4°C. The supernatant was centrifuged at approximately 116,000 x g (50,000 rpm) for 30 min at 4°C. The supernatant was discarded and the microsomal pellet was resuspended in ice-cold 1.15% KCl. The resuspended pellet was centrifuged at 116,000 x g as described above. The supernatant was discarded and the pellet was resuspended in 20 mM Tris-HCl (pH 7.4) containing 250 mM sucrose and 5.4 mM EDTA for a concentration of approximately 1 g wet weight of liver per ml. The microsomal suspension was stored at 80°C.
Protein determination. Microsomal protein was measured by a modified method of Lowry et al. (1951) using bovine serum albumin as standard.
Cytochrome P450 determination. Cytochrome P450 was measured by the carbon monoxidereduced difference spectrum in microsomes (Omura and Sato, 1964), using a Varian Cary 219 spectrophotometer (Varian Inc., Palo Alto, CA). The concentration of cytochrome P450 was calculated using an extinction coefficient of 91 mM1cm1.
p-Nitrophenol hydroxylase assay. 4-Nitrophenol hydroxylase was measured by the method for assay of 4-nitrocatechol reported by Reinke and Moyer (1985), with modifications by Koop (1986)
.
Testosterone hydroxylase. The determination of testosterone hydroxylase activities was conducted by the separation and quantification of hydroxytestosterone metabolites using the method of Purdon and Lehman-McKeeman (1997), modified from van der Hoeven (1984)
. Briefly, samples were diluted to a protein concentration of 0.2 mg/ml and preincubated at 37°C for 3 min in 0.05 M phosphate buffer, containing EDTA, magnesium chloride, glucose-6-phosphate dehydrogenase, glucose-6-phosphate, and NADPH. After preincubation, 10 µl of a solution of testosterone (25 mM in methanol) was added, and incubation was continued for 10 min. Methylene chloride (6 ml) was added, and 1 nmol of cortexolone was added as an internal standard. The sample was vortex-mixed, centrifuged, and the methylene chloride layer was removed and evaporated under nitrogen. The residue was redissolved in 50 µl methanol, to which 100 µl of water was added; then, 100 µl of sample was injected on HPLC for analysis. Hydroxytestosterone metabolites were analyzed by HPLC on a Hewlett Packard 1100 LC Chemstation HPLC System consisting of a binary pump with diode array detector and thermostatted autosampler. A Supelcosil LC-18 column (0.46 x 15 cm, 3 µm; Supelco, Bellefonte, PA) with Supelguard LC-18 guard cartridge (2 cm) was used. Analytes were detected at 247 nm. The solvent system consisted of (A) water and (B) methanol (J.T. Baker, Phillipsburg, NJ). The starting solvent condition was 10% B, and the metabolites were eluted using a linear gradient from 10 to 60% B over 30 min. At 30 min, the concentration of B was held at 60% for 6 min before flushing the column at 90% B for 2 min and then re-equilibration at starting conditions. The flow rate was constant at 1.5 ml/min. To quantify the hydroxytestosterone metabolites, an internal standard method was used. Five standards were prepared containing 1, 2, 3, 4, or 5 nmol of testosterone and 1 nmol of cortexolone in a final volume of 150 µl. The standard solutions were analyzed and a calibration curve of the area ratio of testosterone/cortexolone was constructed. All metabolites were taken to have the same molar response as testosterone and were, therefore, quantified based on the molar concentration calculated from this curve. Results are calculated as rates (pmol/min/mg). The oxidized metabolites of testosterone were identified by comparison with the retention time of authentic standards for the following: 6
, 6 ß, 7
, 2
, 2 ß, 16
, and 16 ß testosterone and androstenedione.
Western blotting. Protein levels of CYP 2E1 were assessed by using isoform-specific antibodies by Western blot; 8 µg of protein from microsomes from animals exposed for 2, 6, and 24 h at all exposure levels were applied to a BioRad Criterion 10% Tris-HCL gel. Expressed rat CYP 2E1 microsomes (Gentest M115r, Woburn, MA) were used as standards. Primary antibody for antirat CYP 2E1 (Gentest 299216) was used with a secondary antibody, donkey antigoat IgG-HRP (sc-2033; Santa Cruz Biotechnology, Santa Cruz, CA). We conducted SDS polyacrylamide gel electrophoresis to separate the microsomal proteins according to molecular weight. Next, we transferred the separated proteins to a nitrocellulose membrane and used the chemiluminescent substrate SuperSignal West Pico (Pierce, Rockford, IL). Spot densitometry on an Alpha Innotech Fluorochem 8000 (San Leandro, CA) was used to obtain an integrated density value (IDV) for each spot.
Statistical analysis. For comparison of cytochrome P450 and monooxygenase activities of control and treatment groups, ANOVA was conducted using Instat version 2.01 (Graphpad Software, San Diego, CA).
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RESULTS |
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For the 1000-ppm exposure and its control groups, the data on total liver microsomal cytochrome P450 are presented in Table 2. The mean levels of total cytochrome P450 measured in the control groups varied between 0.61 and 0.71 nmol/mg microsomal protein. In the rats exposed to ethylene, cytochrome P450 significantly decreased from the control value (6 h) at 5 min and 1, 2, and 12 h. At 10, 15, and 30 min and 4 and 6 h, the mean values were at or below that of the lowest mean control value but were not significantly decreased. At 12 h after the start of exposure, cytochrome P450 was significantly below that of the 12 h control, but at 24 and 48 h the levels were not different from control values. The lowest value observed in the ethylene-exposed rats was 0.53 nmol/mg protein at 5 min, 1 h, and 2 h of exposure, amounting to a decrease of approximately 13% compared with control.
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The activities of other specific cytochrome P450 isozymes were assessed using testosterone as a substrate in rats exposed to 1000 ppm (Table 2). The activity of CYP 2C11 was assessed by the measurement of 2-testosterone hydroxylase. No decreases were observed in 2
-hydroxylase activity, and at a single time point an increase in activity was noted.
The activity of CYP 2A1/2A2 was assessed by the measurement of 7-testosterone hydroxylase. No consistent decreases were observed in the 7
-hydroxylase activity (Table 2). A statistically significant decrease (to 76% of the control activity) was observed at a single time point (5 min).
The activity of CYP 2B1 was assessed by the measurement of 16ß-testosterone hydroxylase. A slight decrease in 16ß-testosterone hydroxylase was observed at some time points during the exposure, but overall a consistent decrease was not observed (Table 2).
In the exposures to 300 and 600 ppm ethylene, liver samples were collected at only 2, 4, and 6 h during exposure and at 12 and 24 h after the initiation of exposure. The mean liver microsomal cytochrome P450 levels in groups exposed to air ranged from 0.740.78 nmol/mg protein (Table 3). Significant decreases in the amount of cytochrome P450 were observed at 2 (600 ppm), 4 (300 and 600 ppm), and 6 h (300 and 600 ppm). The maximal decrease (21%) was observed after exposure for 6 h to 300 ppm ethylene.
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CYP 2E1 microsomal protein levels were assessed by Western blotting (data not shown). At 2 and 24 h, IDVs were slightly lower in all exposure concentrations compared to controls (data not shown). At 6 h, IDVs were approximately 50% of the control for all exposure levels, suggesting a loss of CYP 2E1 protein correlating with a loss of activity. There was no difference in IDV for the groups exposed to ethylene at any of the time points examined.
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DISCUSSION |
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The range of blood concentrations of EO observed in this study (0.060.13 µg/g) was considerably lower than that (0.63 µg/g) reported by Maples and Dahl (1993). The reason for this difference is not known. There are many factors, such as animal source, diet, method of exposure-atmosphere generation, and sample analysis, that could contribute to the differences observed. In this study, we used headspace analysis with GC and FID and standardization based on added EO for the quantitation of EO in blood. Maples and Dahl (1993)
used cryogenic distillation for sample preparation, GC-MS for analysis of the trapped sample containing EO, and EO-d4 added as internal standard. Thus, there are numerous analytical differences between the two studies. The difference in outcome may result from the use of low-resolution GC-MS for the analysis of EO. The molecular ion of EO has the same mass as CO2 (m/z 44). High-resolution GC-MS can be used to monitor this ion at m/z 44.0262 and the corresponding ion for EO-d4 at m/z 48.0513 (Brown et al., 1998
), while resolving EO from CO2 at m/z 43.9898. An alternative approach is to monitor an ion other than m/z 44 to avoid interference from CO2. A GC-MS with a low-resolution mass selective detector was used to monitor ions at m/z 42 for EO and m/z 46 for EO-d4 (Maples and Dahl, 1993
). These ions correspond to different fragments of EO (M+H2) and EO-d4 (M+D). Without careful standardization, comparison of the peak areas of EO with the deuterated internal standard could produce a systematic error in quantitation.
Monooxygenase activities measured in this study were selected to provide an indication of activity for the major CYP isozymes in control rat liver microsomes: testoterone 2-hydroxylation for 2C11, 7
-hydroxylation for 2A1/2A2, 16ß-hydroxylation for 2B1, and 4-nitrophenol hydroxylase for 2E1. Of the various monooxygenase activities measured, 4-nitrophenol hydroxylase was the one most consistently altered, with inhibition reaching its greatest extent at 2 h of exposure to 1000 ppm ethylene. However, 4-nitrophenol hydroxylase activity recovered to control levels by 12 h after the start of the exposure. CYP 2E1 appears to be the major isoform inhibited by exposure to ethylene. As indicated by semiquantitative Western blotting, CYP 2E1 microsomal protein levels also decreased during exposure. The decrease in activity is consistent with the production of a reactive metabolite, modification of the heme of cytochrome P450, and inhibition of activity (Ortiz de Montellano and Mico, 1980
; Ortiz de Montellano et al., 1981
). The recovery of CYP 2E1 activity is consistent with the rapid removal of ethylene following exposure and the rapid resynthesis of CYP 2E1. Treatment of rats with inhibitors of CYP 2E1 can result in rapid increases in activity following inhibition. After administration of trans-dichloroethylene (100 mg/kg, ip) to rats, hepatic CYP 2E1 activity, measured by 4-nitrophenol hydroxylase, was profoundly inhibited at 2, 5, and 12 h after dosing; but, by 24 h, this activity had returned to control levels (Mathews et al., 1998
).
The inhibition of activity for CYP 2E1 and metabolism of ethylene to EO results in a supralinear dose-response curve (Fig. 3) for AUC06 h plotted against exposure concentration (i.e., a greater response per unit dose at low-exposure concentration compared with higher exposure concentrations). The AUC06 h for unexposed rats was calculated to be 1.44 nmol.h/l from the estimated EO concentration in blood from endogenous ethylene production of 0.24 nmol/l (Csanady et al., 2000). At 1000 ppm ethylene, the AUC06 h was calculated as 14.0 µmol.h/l. This compares with a calculated value for EO exposure at 100 ppm in male rats of 142.7 µmol.h/l (Fennell and Brown, 2001
), suggesting that a 1000-ppm exposure to ethylene produces EO blood AUC similar to a 10-ppm exposure to EO. Others have suggested that exposure to 1000 ppm ethylene is equivalent to exposure to 2.2 ppm EO (Csanady et al., 2000
) and 5.6 ppm EO (Bolt and Filser, 1987
), based on pharmacokinetic models, or 4.411.3 ppm EO, based on the formation of 7-(2-hydroxyethyl)guanine in DNA from liver, spleen, brain, and lung (Walker et al., 2000
).
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At all three concentrations of ethylene examined in this study (300, 600, and 1000 ppm), the inhibition of CYP 2E1 activity was observed. All of the exposure concentrations used were above the estimated Km values for ethylene metabolism (Andersen et al., 1980; Bolt and Filser, 1987
). It is not clear whether the inhibition would be observed at lower exposure concentrations, particularly those encountered with human exposures in the workplace. CYP 2E1 is involved in the metabolism of a variety of chemicals (Guengerich et al., 1991
). Interference with the metabolism of other CYP 2E1 substrates is a possible concern. This study focused on the effect of ethylene exposure on liver microsomal CYP 2E1. Ethylene may also interact with CYP 2E1 in other tissues.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Little 176, RTI International, 3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park, NC 27709-2194. Fax: (919) 541-6499. E-mail: fennell{at}rti.org.
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