E.I. du Pont de Nemours and Company, Haskell Laboratory for Health and Environmental Sciences, PO Box 50, 1090 Elkton Road, Newark, Delaware 19711
Received October 18, 2003; accepted January 26, 2004
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ABSTRACT |
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Key Words: 2-chloro-1,3-butadiene; microsomes; cytosol; liver; lung; mouse; rat; hamster; human; in vitro kinetics.
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INTRODUCTION |
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The need to understand possible adverse health effects in humans has led to extensive evaluation of CD in experimental animals. Recent literature reviews have described the acute, subchronic, and chronic toxicity (Melnick and Sills, 2001; Valentine and Himmelstein, 2001
). The most significant finding was CD-induced tumorigenicity in F344/N rats and B6C3F1 mice exposed to
80 ppm for 2 years (Melnick et al., 1999
, 1996
; NTP 1998
). Tumors in Fischer rats included the lung, oral cavity, thyroid gland, kidney, and mammary gland. Mouse tumors were observed in the lung, circulatory system, Harderian gland, forestomach, kidney, mammary gland, skin, mesentery, Zymbal gland, and liver. In contrast, no tumors were observed in Syrian hamsters. A weak response in mammary tissue was observed in female Wistar rats exposed to 10 or 50 ppm CD for up to 2 years (Trochimowicz et al., 1998
).
In vitro genotoxicity studies revealed that CD is mutagenic, primarily in bacterial reverse mutation assays with metabolic activation, while other in vitro and in vivo assays for either gene mutation or structural chromosomal damage were negative (Drevon and Kuroki, 1979; Foureman et al., 1994
; NTP, 1998
; Tice, 1988
; Tice et al., 1988
; Vogel, 1979
). A recent study showed that a known CD metabolite, racemic (1-chloroethenyl)oxirane (1-CEO), was mutagenic in three Salmonella typhimurium strains (Himmelstein et al., 2001b
).
The in vitro biotransformation of CD has been studied to identify metabolites that might contribute to toxicity or mutagenicity (Bartsch et al., 1979). More recently, the microsomal metabolism of CD to 1-CEO was confirmed as a cytochrome P450-dependent reaction (Himmelstein et al., 2001a
), and the S- and R-enantiomers of 1-CEO were identified (Cottrell et al., 2001
). Enzyme-mediated hydrolysis of 1-CEO occurred by microsomal epoxide hydrolase (Cottrell et al., 2001
; Himmelstein et al., 2001a
). Conjugation of 1-CEO with GSH has been shown in liver cytosolic fractions (Himmelstein et al., 2001a
), consistent with earlier in vivo studies showing depletion of nonprotein sulfhydryls and the presence of thioethers in urine of rodents exposed to CD (Jaeger et al., 1975
; Plugge and Jaeger, 1979
; Summer and Greim, 1980
).
The goal of this research was to quantify the in vitro rates of CD-related biotransformation reactions in liver and lung tissue fractions of the rodent strains tested in the cancer bioassays and humans. Liver and lung were studied, because these tissues were prone to tumor induction and represented tissues important to xenobiotic metabolism. The simultaneous time course of cytochrome P450-dependent oxidation of CD was compared with rates of 1-CEO formation and hydrolysis, using a two-compartment kinetic model to account for the volatility of both analytes.
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MATERIALS AND METHODS |
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Source of microsomes and cytosol.
Rodent liver microsomes and cytosol were purchased from two suppliers (In Vitro Technologies, Baltimore, MD or XenoTech, Lenexa, KS). Both suppliers used differential centrifugation as the method of preparation. Microsomes and cytosol were prepared from pooled livers from male B6C3F1 mice, Fischer F344 rats, Wistar rats, or Golden Syrian hamsters obtained from Charles River Laboratories (Raleigh, NC). Rodent lung microsomes and cytosol were prepared in-house using animals obtained from Charles River Laboratories. The fractions were prepared by differential centrifugation as previously described (Csanády et al., 1992; Guengerich, 1994
). All fractions were stored at <-70°C.
Human tissue fractions were also prepared by differential centrifugation. For experiments involving hydrolysis of 1-CEO, pooled liver microsomes from 15 individuals were used (Lot #1032, In Vitro Technologies). Experiments involving simultaneous CD and 1-CEO measurements used pooled liver microsomes from 10 individuals (Lot# 0010186, H1000, XenoTech). Human lung microsomes were obtained from a pool of five individuals (Cat # HPM 501, Lot 1.0, Human Biologics International, Scottsdale, AZ). Human liver cytosol was obtained from a mixed pool of 15 individuals (Lot# HHC 280, Tissue Transformation Technologies, Edison, NJ). Human lung cytosol was from a single male (Lot # HG714100, Tissue Transformation Technologies). Human donor demographic information and metabolic characterization is available but not reported here for the sake of brevity.
Microsomal hydrolysis of 1-CEO.
The time course of 1-CEO metabolism was measured in liver and lung microsomes from each species by further refinement of the method described in Himmelstein et al. (2001a). Vials (1 ml liquid/10 ml vial) were prepared with 0.1 M phosphate buffer (pH 7.4), MgCl2, (15 mM), EDTA (0.1 mM), glucose-6-phosphate (0.8 mM), and glucose-6-phosphate dehydrogenase (1 U/ml). The vials were sealed with crimp top, Teflon®-coated silicone septa (Gerstel US, Baltimore MD) with a ParafilmTM sheet between the metal crimp top and septa and plastic tape around the outside rim of the crimp top to insure a positive seal. Incubations were performed using a programmable x-y-z robotic multi-purpose gas chromatograph (GC) sampler (MPS2, Gerstel, Baltimore, MD). Sample vials were transferred to the MPS2 agitator-heater block and preheated for 11 min at 500 rpm. Known concentrations of 1-CEO (prepared in Tedlar bags) were added by manually flushing the vial headspace with approximately 15 ml of vapor (see Fig. legends for actual concentrations used). The headspace concentration was immediately quantified by robotic transfer from the vial to the GC inlet, using the MPS2 1.0 ml gas-tight syringe. The optimal syringe temperature (38°C), sample injection volume (200 µ;l), and injection speed (800 µ;l/s) were pre-determined experimentally. The protein concentration (0.253 mg/ml) and duration of incubation (060 min) were also optimized during preliminary experiments. Microsomal protein for the definitive experiments (1050 µ;l to final concentration of 0.5 mg/ml) was manually added to the vial to start the enzymatic time course reactions. Between injections, the headspace-sampling syringe was flushed with helium (at 48.5°C for 3 min) to clear residual chemical. Prior to the next sample (t = 12 min), an injection of air was made to check for residual chemical, which if present, was subtracted from the next headspace sample. The air, sample injections, and syringe flushes were repeated (t = 12, 24, 36, 48, and 60 min) to determine the time course of metabolism. Control incubations included phosphate buffer or heat-inactivated microsomes. The concentration of 1-CEO in vial headspace was calculated using linear regression analysis from gasbag standards. Headspace GC-mass spectrometry (GC/MS) analysis by single ion monitoring (m/z 39/1-CEO) was conducted as described previously (Himmelstein et al., 2001a
). The limit of quantification of 1-CEO (and CD in subsequent experiments described below) was 0.001 nmol/ml of headspace (equivalent to 24 ppb).
Microsomal oxidation of 1-CEO.
Vials were prepared with incubation reagents as described above. Cofactor NADP+ was added to the vial 10 s before the addition of microsomal protein. Time-course data for 1-CEO, with and without NADP+, were measured over a range of starting 1-CEO concentrations. In mouse liver microsomes, the only species to show significant metabolism, additional incubations were conducted in the presence of 4-methylpyrazole (4-MP) or 1-aminobenzotriazole (ABT) as inhibitors of cytochrome P450-dependent oxidation (Halpert et al., 1994). Stock solutions (100 mM) of 4-MP in phosphate buffer or ABT in acetonitrile were added to a final concentration of 100 µ;M; control incubation mixtures included equal amounts of buffer or acetonitrile. Additional incubations were conducted to isolate the potential oxidative metabolite of 1-CEO. Incubation mixtures containing mouse liver microsomes were agitated in a Bucher shaker (Labconco, Lenexa, KS) at 37°C with 1-CEO (10 mM) in 10 ml total volume in sealed 24 ml vials, with or without NADP+. After 30 min, the reactions were stopped by the addition of cold ethyl acetate (10 ml) and NaCl (5 g/vial). Following centrifugation (1700 x g for 5 min) and extraction with ethyl acetate (10 ml, then 5 ml), the solvent was analyzed by full scan GC/MS. Instrument conditions were similar to those used previously (Cottrell et al., 2001
).
Cytosolic GSH conjugation of CD or 1-CEO.
Preliminary experiments showed no reactivity of CD with GSH in cytosol or heat-inactivated cytosol. For 1-CEO, the incubation vials were prepared as described above for the microsomal 1-CEO hydrolysis experiments, except that cytosolic protein from each species (12 mg protein/ml) and GSH (10 mM) were used. Headspace samples (400 µ;l) were taken at 0, 12, 24, 36, 48, and 60 min, after the reactions were started by the addition of cytosol and GSH. Control incubations in phosphate buffer were conducted using 1-CEO alone, 1-CEO + GSH, and 1-CEO + GSH + heat-inactivated cytosol.
Microsomal oxidation of CD.
The time course of CD disappearance and 1-CEO formation and hydrolysis was measured simultaneously in liver or lung microsomes. For liver microsomal incubations from each species, vials were prepared with the incubation reagents described above for microsomal 1-CEO hydrolysis. After preincubation (37°C for 5 min), an equal volume of vial headspace was removed from the vial and replaced with known concentrations of CD vapor. The vial was equilibrated for approximately 10 min, and reactions were started by the addition of microsomal protein (1 mg/ml) and NADP+ (0.4 mg/ml of suspension). The protein concentration was optimized during preliminary experiments (0.53.0 mg/ml). Control incubations were performed without NADP+ or with NADP+ and heat-inactivated microsomes. Incubation start times were staggered, and up to five vials per exposure concentration were used to describe the time course of headspace 1-CEO relative to the disappearance of CD from the headspace. The repeated headspace samples (400 µ;l, 46 injections per vial) were analyzed at 12-min intervals for up to 1 h by GC/MS. For lung microsomes, the incubation conditions were identical to those used for liver microsomes except for the different starting CD concentrations. For some incubations, 4-MP or ABT (at final concentrations of 100 µ;M) were added to determine if CD oxidation was dependent on cytochrome P450-monooxygenase.
Model development.
A two-compartment model was used to describe the distribution and metabolism of CD and 1-CEO in the closed vial system. Mass transport between the air and liquid phases was assumed to be instantaneous, and thus partition coefficients were used to describe distribution between phases. The liquid-to-air volume ratio (1-to-10) was small, and the liquid was stirred vigorously to ensure rapid mixing during preincubation and reaction periods (described above). A known volume of air containing a fraction of the air phase CD and 1-CEO was removed from the system at each sampling time. Experiments with PBS buffer or heat-inactivated microsomes were used to calculate the control loss from the vial system. The measured concentrations of CD and 1-CEO in the air phase and their known liquid-to-air partition coefficients (0.69 for CD and 57.9 for 1-CEO; Himmelstein et al., 2001a) were used to calculate the liquid phase concentrations. Given the control loss, the model was used to calculate the rate constants for 1-CEO hydrolysis, further 1-CEO oxidation if present, CD oxidation, and GSH conjugation. Microsomal 1-CEO hydrolysis and total CD oxidation were parameterized as Michaelis-Menten-type equations. The 1-CEO formed by CD oxidation was parameterized, initially using the constants for total CD oxidation and the 1-CEO hydrolysis constants previously calculated. For cases where 1-CEO concentrations were over-predicted, the model predictions were reduced by dividing the total CD oxidation into two pathways; one parameterized from the 1-CEO formation, and the other from total CD disappearance, representing metabolism to uncharacterized products. Disappearance of 1-CEO from the headspace was used to quantify the conjugation of 1-CEO with cytosolic GSH. Non-enzymatic conjugation was simulated as a first-order reaction and enzymatic conjugation was simulated as a pseudo second-order process involving GST-mediated conjugation of 1-CEO with GSH and binding of 1-CEO directly with the enzyme.
The differential equations describing the model, along with the detailed nomenclature, are given in the appendix (see Supplementary Data). Model simulation was conducted using the Advanced Continuous Simulation Language (ACSL version 11.8.4, Aegis Technologies Group, Huntsville, AL). Parameter optimization was conducted using ACSL Math and Optimize version 2.5.4 (Aegis Technologies Group). All of the model metabolic parameters were optimized in ACSL-Optimize, using the Nelder-Mead method and a relative error minimization-based, log-likelihood function. For pathways with more than one metabolic constant, the parameters were optimized simultaneously to avoid local log-likelihood minima. The model code is available upon request.
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RESULTS |
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DISCUSSION |
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Oxidative Metabolism of CD
CD metabolism by liver microsomes was similar across species with intrinsic clearance showing a maximum difference of about two-fold greater in the mouse than the human. The results were consistent with recent findings for CD oxidation in microsomal liver preparations with mice > rats > humans (Munter et al., 2003). Comparison of results for CD and 1,3-butadiene (a nonchlorinated chemical analog) is possible given the similar experimental approach. The Vmax/Km values for microsomal CD oxidation appear to be faster than those reported for the oxidation of butadiene to epoxybutene (Csanády et al., 1992
). For example, the range of maximum rates (µ;mol/h/mg protein) for CD (0.068 to 0.29) was similar to butadiene (0.04 to 0.16). However, the corresponding apparent Km constants for CD (0.53 to 1.33 µ;M) suggest slightly greater affinity of CD for cytochrome P450 compared with butadiene (2 to 5.1 µ;M). The Vmax/Km for butadiene in liver microsomes was mouse (78) > human (14) > rat (9), all lower compared with the Vmax/Km values for CD (Table 1).
Oxidation rates for CD and butadiene also were comparable for lung microsomes. The Vmax values for CD and butadiene were 0.1- and 0.14-µ;mol/h/mg protein, respectively; the corresponding apparent Km values were 1.5 and 5 µ;M. The intrinsic clearance for total CD oxidation was greater for the mouse (66.7) when compared to the other species (1.3). For butadiene, the Vmax/Km values in the lung microsomes were mouse (28) > human (4.5) rat (1.2). The differences in the Vmax/Km values for CD and BD in the mouse appear consistent with the relatively greater potency of CD than butadiene for lung tumors, although species sensitivity differences may also be due to tissue concentration differences caused by chemical partitioning. For example, the lowest exposure concentration that produced lung tumors in male mice was 12.8 ppm CD compared with 62.5 ppm for butadiene (Melnick et al., 1999
).
Microsomal Hydrolysis of 1-CEO
The liquid-to-air partition coefficient for 1-CEO (58:1) used for predicting the rate of metabolism in microsomal suspensions was similar to oxirane (77:1) (Schmiedel et al., 1983). The Vmax/Km for 1-CEO hydrolysis in liver microsomes indicated that mice were about two times slower than rats and six times slower than hamsters or humans. The apparent Km values were consistent with those published for other oxirane analogs such epoxybutene, oxirane, and propylene oxide, although there appear to be differences in Vmax for these different substrates (Csanády et al., 1992
; Brown et al., 1996
; Faller et al., 2001
; Krause et al., 1997
; Kreuzer et al., 1991
). A previous study also established that 1,1,1-trichloropropene oxide (TCPO), a competitive substrate of epoxide hydrolase, inhibited this reaction (Himmelstein et al., 2001a
). The product formed in the absence of inhibition was 3-chloro-3-butene-1,2-diol (Cottrell et al., 2001
).
Microsomal Oxidation of 1-CEO
This reaction in liver microsomes appeared to be limited to mouse, and was not evident in liver microsomes from human, rat, or hamster. In the case of 1,3-butadiene metabolism, a faster rate of epoxybutene oxidation to diepoxybutane has been observed in mouse liver microsomes compared with the rat or human (Csanády et al., 1992). The intrinsic clearance of 1-CEO by oxidation in mouse liver microsomes (23 ml/h/mg protein) was approximately 10-fold less than the clearance of CD oxidation (224 ml/h/mg protein). It is not yet known if oxidative metabolism of 1-CEO occurs in mouse lung. The dependence of the reaction on NADP+ and inhibition by 4-MP or ABT clearly suggests involvement of cytochrome P450 monooxygenase. Oxidation of 1-CEO by the mouse potentially could lead to a reactive intermediate such as a diepoxide, but our results (data not shown) confirmed the diol hydrolysis product of 1-CEO previously reported (Cottrell et al., 2001
). Cottrell et al. (2001)
reported that a synthetic standard of chloroprene diepoxide was chemically unstable in aqueous media, forming products that have yet to be characterized.
Kinetics of CD and 1-CEO
To our knowledge, this is the first example of simultaneous time-course analysis of a parent chemical and its metabolite, either for CD or for similar chemical analogs. The 1-CEO time course initially was modeled as if it was the sole product of CD, but this approach indicated that 1-CEO formation with subsequent hydrolysis represented a fraction of the total CD oxidation occurring in vitro. Because 2-CEO was unstable in aqueous media and could not be quantified, a determination of the mass balance between the two epoxide-formation pathways was not possible. However, a satisfactory description of the 1-CEO time course data was obtained by modeling a split of the cytochrome P450-dependent CD oxidative metabolite leading to 1-CEO and other uncharacterized product(s), while using the measured 1-CEO epoxide hydrolase kinetics to account for 1-CEO elimination (Fig. 5). The formation of 1-CEO was best described as representing 25% of the total oxidative metabolism of CD across the animals tested (Table 3). Results similar to those presented in Figure 5 were also obtained by re-optimizing the rates of 1-CEO hydrolysis so that the rate of 1-CEO removal was substantially increased. This alternative model description was considered inappropriate because 1-CEO epoxide hydrolase activity was experimentally measured. The finding that the majority of CD oxidation leads to metabolites other than 1-CEO warrants further investigation as these metabolites may be important to understanding relative tumorigenicity across species.
Adjustment of 1-CEO formation as a proportion of total CD oxidation in lung microsomes was less robust than that for the liver because of the limited amount of 1-CEO time course data (Fig. 6). In rodents, estimates of adjusted Vmax/Km were 322% of the total oxidative CD metabolism (Table 3). The value of 78% for human lung microsomes likely represents an overestimation because of rapid removal of 1-CEO by epoxide hydrolase. An inherent limitation of estimating in vitro metabolism using microsomes prepared from whole lungs is the distribution of different cell types and low enzyme activity in the homogenized lung tissue (Plopper et al., 1991, Seaton et al., 1996
). Despite this shortcoming, the greater CD oxidation and 1-CEO formation in the mouse compared with the other rodents provides some insight regarding the greater tumor response of this tissue in the B6C3F1 mouse.
Cytosolic Metabolism of 1-CEO
The experimental approach was similar to that used to describe GST-mediated metabolism of 1,2-epoxy-3-butene and propylene oxide (Faller et al., 2001; Kreuzer et al., 1991
). The half-life for the spontaneous first-order reaction between 1-CEO and GSH was approximately 10 h. This appears to be longer than the half-lives for spontaneous GSH reactivity with propylene oxide of 1.7 h (Faller et al., 2001
), and epoxybutene (3.9 h, Csanády et al., 1992
; 0.8 h, Kreuzer et al., 1991
). The enzymatic pseudo-second order reaction between 1-CEO and GSH suggests greater GST capacity in liver than lung. Enzymatic GSH conjugation with 1-CEO was first thought to be a standard saturable Michaelis-Menten reaction but a closer analysis of the data showed divergent behavior. In particular, the conjugation rate at a given concentration was dependent on the starting concentration (Fig. 7D). For example, the rate in hamster liver cytosol for the 4.1 nmol/ml starting 1-CEO headspace concentration was faster than at 41 nmol/ml. Standard Michaelis-Menten kinetics would have shown approximately parallel data. This behavior suggested that active enzymatic sites were being consumed rather than following standard Michaelis-Menten kinetics. The description of this enzymatic reaction as pseudo second order is consistent with the known irreversible binding of substrate with some forms of GST (Raham et al., 1999
). Numerous other epoxides, such as oxirane, propylene oxide, epoxybutene, diepoxybutane, also undergo metabolism by GST (Brown et al., 1996
; Faller et al., 2001
; review by Himmelstein et al., 1997
). Additional research is needed to identify the structures of the GSH conjugates and the potential impact of GSH depletion on in vivo toxicity.
Conclusions
Total CD oxidation was evident in liver and lung microsomal tissue fractions. Species and tissue differences were observed, the most dramatic of which was a faster rate of CD metabolism in the mouse lung compared with the other species. The time course data for simultaneous microsomal metabolism of CD and 1-CEO were explained by formation of 1-CEO and uncharacterized products; across species 1-CEO formation in the liver fraction appeared to be 25% of the total CD oxidation. The findings support the need for better understanding of the kinetic relationship between oxidative formation of epoxide metabolites and their elimination by epoxide hydrolase for analogs such as butadiene, ß-chloroprene, and isoprene. Cytosolic GST-mediated conjugation was quantified as a pathway for 1-CEO metabolism. Overall, the in vitro kinetic parameters developed here improve the understanding of CD metabolism. The kinetics of total CD oxidation measured in liver and lung microsomes have been used to develop a physiologically based toxicokinetic model to aid in CD risk assessment (Himmelstein et al., 2004).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (302) 366-5003. E-mail: matthew.w.himmelstein{at}usa.dupont.com
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