Mechanistic Aspects of the Metabolism of 1, 3-Dichloropropene in

route of administration upon DCPO formation. The amounts of DCPO present in the liver and blood of rats and mice were measured following oral (po) and...
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Chem. Res. Toxicol. 2000, 13, 1096-1102

Mechanistic Aspects of the Metabolism of 1,3-Dichloropropene in Rats and Mice Michael J. Bartels,* Kathy A. Brzak, Alan L. Mendrala, and William T. Stott Health and Environmental Research Laboratory, 1803 Building, The Dow Chemical Company, Midland, Michigan 48674 Received June 12, 2000

1,3-Dichloropropene (DCP) is used in agriculture for the control of nematodes in a variety of food crops. The major routes of metabolism for this halogenated aliphatic compound involve conjugation with glutathione and oxidation to carbon dioxide. An additional, minor route of metabolism proposed for this compound involves epoxidation to the corresponding 1,3dichloropropene oxide (DCPO). Recent in vivo studies have provided evidence for the formation of DCPO in mice following intraperitoneal (ip) administration of 350-700 mg of DCP/kg, which is equal to, or exceeds, the reported oral LD50 for this compound in mice [Schneider, M., et al. (1998) Chem. Res. Toxicol. 11, 1137-1144]. The potential for epoxidation of DCP in rats and mice at lower doses administered orally was therefore examined. Following oral administration of 100 mg of DCP/kg of body weight to F344 rats and B6C3F1 mice, no DCPO was found in the liver or blood 0-90 min postdosing at a relatively low detection limit (10 ng/g of tissue). Only very low levels of DCPO were seen following ip administration of 100 mg of DCP/kg of body weight in blood of B6C3F1 mice. Substantial levels of DCPO were only seen as a metabolite of DCP following ip administration of 700 mg of DCP/kg to B6C3F1 or Swiss-Webster mice. Significant nonlinearity of DCP epoxidation was evident following ip administration, with approximately 130-fold less DCPO in mice given 100 vs 700 mg/kg. The time course of DCPO formation could only be followed for 76 min, due to 100% mortality in Swiss-Webster mice at the 700 mg/kg dose level. The formation of measurable DCPO in mice was also accompanied by acute hepatic damage following ip administration of 100 or 700 mg of DCP/kg to mice. In contrast, no evidence of acute toxicity was noted in mice treated with 100 mg/kg via oral gavage. These data suggest that measurable epoxidation of DCP to DCPO, in the rodent, occurs only at relatively high dose levels which result in acute hepatic injury or death. It was concluded that findings of DCPO formation at lethal doses administered via bolus internal injections do not reflect DCPO formation at lower doses administered via the natural portal of entry.

Introduction 1,3-Dichloropropene (DCP) is used in agriculture for the control of nematodes in a variety of food crops (1). DCP has been shown to be highly metabolized in mice and rats as well as in human volunteers. The majority of an oral dose given to rats or mice has been shown to be rapidly eliminated in the urine, primarily as mercapturic acid conjugates of the cis and trans isomers (2-5). Glutathione conjugation has been also shown to be a significant route of metabolism of DCP in humans, affording the two mercapturic acid isomers (6-8). Glutathione conjugation of this compound is generally thought to be a detoxifying metabolic pathway. Formation of carbon dioxide from DCP is also a significant route of metabolism in rats and mice, representing as much as 24% of the administered dose (3, 9). A third metabolic pathway has been proposed by several authors, involving initial epoxidation of DCP, to afford a potentially mutagenic metabolite, 1,3-dichloro1-propene oxide (DCPO) (10, 11). Watson et al. provided indirect evidence for the formation of this metabolite in an in vitro mutagenicity assay. Recent studies by * To whom correspondence should be addressed. Phone: (517) 6369057. E-mail: [email protected].

Schneider et al. have provided evidence for epoxidation of DCP in male Swiss-Webster mice. However, these experiments utilized ip-injected doses of 350-700 mg/ kg in a DMSO vehicle, which are equal to, or exceed, the reported oral LD50 for this compound in the CD-1 and JCL:ICR mice and Fischer 344 rats (215-640 mg/kg) (12). This study was undertaken to investigate the potential for epoxidation of DCP in rats and mice at acutely less toxic dose levels and to examine the potential effect of route of administration upon DCPO formation. The amounts of DCPO present in the liver and blood of rats and mice were measured following oral (po) and/or intraperitoneal (ip) administration of DCP. The vehicles used for the po (corn oil) and ip (DMSO) routes of administration were chosen to match the vehicles used in previous po (2) and ip (11) metabolism studies. The effect of dose level, route, and strain on epoxidation of DCP was examined in mice. High-dose (700 mg/kg) experiments were performed in Swiss-Webster mice to replicate the results of Schneider (11). The majority of the work was performed in B6C3F1 mice, the strain generally used in toxicity and/or oncogenicity studies with DCP (13). A low-dose oral experiment was also performed in Fischer 344 mice, which is also the strain used in

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Metabolism of DCP in Rats and Mice

toxicity and/or oncogenicity studies with DCP (13). Tissue samples were prepared frozen to minimize the rapid degradation of DCPO. The time course of DCPO degradation in liver and blood was also examined in rats and mice in vitro. Both DCP and DCPO were assessed via GC/MS, utilizing stable-isotope-labeled internal standards to correct for systematic variability during sample preparation and analysis.

Experimental Procedures Reagents. DCP, a mixture of cis and trans isomers, was obtained from Dow Chemical Co. (Midland, MI). The purity was determined to be 98.2% via GC. This test material was purified via column chromatography on silicic acid, as per Watson et al. (10), to decrease the level of DCPO impurities present in the test material. trans-DCP was obtained from Dow Chemical Co. cis/trans-d4-DCP was obtained from Cambridge Isotope Laboratories (Andover, MA). cis/trans-, trans-, and cis-d4-DCPO were synthesized from the analogous DCP isomers via the method of Kline (14). All other compounds and solvents were reagent grade or better. Test Animals. Male Fischer 344 rats (218-246 g), male B6C3F1 mice (26-30 g), and male albino Swiss-Webster (SW) mice (27-29 g) were purchased from Charles River Breeding Laboratories (Raleigh, NC). Upon receipt, the animals were examined by a veterinarian and found to be in good health. Before use, the animals were acclimated to the laboratory environment for at least 1 week. The rooms in which the animals were housed had a 12 h photocycle and are designed to maintain adequate environmental temperature, relative humidity, and air flow for the mice and rats. Municipal drinking water and Purina Certified Rodent Chow 5002 (Purina Mills, Inc., St. Louis, MO) were provided ad libitum except that food was withdrawn from mice and rats given DCP orally, approximately 2-4 and 16 h before the administration of the test material, respectively. All of the animals were held in standard animals cages both prior to and following dose administration. Dose Administration. DCP, as a solution in corn oil, was administered to male F344 rats and male B6C3F1 mice by gavage, with blunted feeding needles, at a target dose of 100 mg/kg of body weight (dose volume of 5 mL/kg). DCP, as a solution in DMSO, was administered to male B6C3F1 and SW mice, via ip administration, at a target dose of 100 mg/kg (B6C3F1 only) of body weight or 700 mg/kg (both strains) of body weight (dose volume of 5 mL/kg). The DMSO dose solutions of DCP were held at room temperature for at least 4 h prior to administration to eliminate any DCPO impurities present in the test material (11). Sample Collection. At the specified time after dose administration, the test animals were euthanized with CO2 and exsanguinated via cardiac puncture. An aliquot (100 µL) of the blood sample was taken and prepared for clinical chemistry analysis of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST). The remainder of the blood sample (0.05-0.5 g) was immediately dispensed into a tared vial containing ethyl acetate (0.5 mL), d4-DCPO, and d4-DCP (5060 ng each of cis- and trans-d4-DCPO/g of solvent and 11-13 µg each of cis- and trans-d4-DCP/g of solvent). The blood samples were mixed by vortexing (approximately 30 s) and gross blood weights taken. The extracted blood samples were placed in an ice bath until they were centrifuged (below). Liver samples were obtained via dissection, blotted dry, immediately frozen in liquid nitrogen, and crushed in a Bio-Pulverizer manual impact hammer (Biospec Products, Bartlesville, OK). The granularized, frozen liver tissue was scraped into a tared vial containing extraction solvent (ethyl acetate; 12-20 mL for rats and 3-5 mL for mice), saturated saltwater (3-5 mL for rats and 0.5-1 mL for mice), and internal standards (55-80 ng/g of solvent each of cis- and trans-d4-DCPO and/or 12-14 µg/g of solvent each of cis- and trans-d4-DCP). The liver samples were mixed by

Chem. Res. Toxicol., Vol. 13, No. 11, 2000 1097 vortexing (approximately 30 s), gross weights taken, and the samples placed in an ice bath until they were centrifuged. The blood and liver samples were centrifuged (10 min at 12501640g). The ethyl acetate extracts were then analyzed via NCIGC/MS for the quantitation of the cis and trans isomers of both DCPO and DCP. The time of sample collection for blood was documented as the actual time elapsed between dose administration and extraction of the blood sample. The time of sample collection for the liver sample was documented as the time elapsed between dose administration and freezing of the liver tissue sample in liquid nitrogen. In Vitro Degradation of DCP and DCPO. Blood and liver samples were obtained from control F344 rats and B6C3F1 mice. Livers were perfused with 0.9% saline and homogenized in approximately 3 volumes of 0.1 M Tris/0.1 M KCl/0.001 M EDTA/20 µM BHT (butylated hydroxytoluene) buffer (pH 7.4). The homogenized liver samples were centrifuged at 9000g for 30 min. The resulting supernatants were further diluted 10and 100-fold with Tris buffer (above). A weighed aliquot of freshly isolated blood (3-4 g for rats and 1.7 g for mice) or diluted liver homogenate (5 g) from control animals was fortified with DCPO at approximately 300 ng/g each of cis- and transDCPO. The temperature of the solutions was maintained at 37 °C, and at specified times (0.25, 1, 2, 5, or 10 min), an aliquot (0.4-1 mL) was removed and immediately placed in a vial containing ethyl acetate and the internal standards (50-70 ng/g solvent each of cis- and trans-d4-DCPO). Time-zero samples were prepared by adding the DCPO directly to the extraction solvent, followed by the addition of control blood or liver homogenate. Sample Analysis. ALT and ASP levels were determined in the serum samples on a Boehringer Mannheim/Hitachi model 914 automated clinical analyzer (Indianapolis, IN). Analysis of DCP in blood and liver extracts was performed with a Hewlett-Packard HP 5890 Series II gas chromatograph (San Jose, CA) and a Finnigan SSQ-710 mass spectrometer (San Jose, CA) with the following representative conditions. Separation was performed on a J&W DB-5MS capillary column (30 m × 0.25 mm i.d., 0.5 µm film thickness; J&W Scientific, Folsom, CA) with helium carrier gas at a head pressure of 10 psi. The injection volume was 1 µL with a 0.5 min splitless injection. The temperature program was 50 °C for 5.0 min to 150 °C at a rate of 15 °C/min, and then to 300 °C at a rate of 30 °C/min. Injector and capillary transfer temperatures were both 175 °C. Mass spectral conditions consisted of negative-ion chemical ionization (NCI) with methane as the reagent gas. The ion source temperature was 150 °C, and the quadrupole temperature was 70 °C. Selected ion monitoring (SIM) of the M - Hparent ions (m/z 109 and 114 at 0.1 s per ion per scan) was employed for quantitation of DCP and d4-DCP isomers. Finnigan GC/MS data files were converted to HP-5973 format with the MassExchange progam (Sierra Analytics, Modesto, CA), and data files were quantitated and prepared as figures with HP5973 Data Analysis software (version A.03.00). Analysis of DCPO in blood and liver extracts was performed with a Hewlett-Packard HP 6890 gas chromatograph and a Hewlett-Packard 5973 mass spectrometer with the following representative conditions. Separation was performed on a J&W DB-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) with helium carrier gas at a head pressure of 7 psi. The injection volume was 2 µL with a 0.3-0.5 min splitless injection. The temperature program was 50 °C for 8.0 min to 300 °C at a rate of 30 °C/min. Injector and capillary transfer temperatures were 175 and 280 °C, respectively. Mass spectral conditions consisted of NCI with methane as the reagent gas. The ion source temperature was 275 °C, and the quadrupole temperature was 125 °C. SIM of the Cl- fragment ions (m/z 35 and 37 at 0.125 s per ion per scan) was used for quantitation of DCPO and d4-DCPO isomers. Analysis of DCPO in in vitro incubations of blood and liver homogenate was performed as described above, with the following exceptions: positive-ion chemical ionization (methane),

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Table 1. In Vitro Degradation of DCPO in Blood and Liver of Rats and Mice degradation half-life (min)a sample matrix

cis-DCPO

trans-DCPO

rat blood rat liver, 10-fold dilution rat liver, 100-fold dilution rat liver, 10-fold dilution, boiled mouse blood mouse liver, 10-fold dilution mouse liver, 100-fold dilution mouse liver, 10-fold dilution, boiled buffer only

1.37 ( 0.13 2.56 ( 1.34 15.70 ( 2.33 18.59 ( 2.70 2.42 ( 0.73 1.89 ( 0.55 15.61 ( 4.45 16.52 ( 0.90 19.54 ( 0.73

1.04 ( 0.15 1.80 ( 1.03 12.43 ( 5.16 20.59 ( 0.73 2.14 ( 0.87 1.04 ( 0.29 9.45 ( 1.14 19.81 ( 0.63 21.77 ( 1.45

a n ) 3-4 per matrix; initial DCPO concentration of approximately 300 ng/mL (each isomer).

SIM of the M - HCl+ fragment ions of DCPO (m/z 91 and 93) and d4-DCPO (m/z 95) at 0.05 s per ion per scan. Statistical Analysis. Pharmacokinetic parameters such as in vitro degradation half-lives and in vivo AUC blood values were calculated with PK Solutions, version 2.0.2 (Summit Research Services, Ashland, OH). AUC values for animals sacrificed within 60 min were determined by extrapolating 60 min concentrations from calculated elimination rates.

Results and Discussion Extraction of DCPO from liver tissue has been reported to be quite efficient, with recoveries of 81-95% (11). In the study presented here, however, much lower recoveries were observed. Fortification of freshly isolated liver tissue with approximately 220 ng of DCPO/g immediately prior to homogenization and extraction with ethyl acetate [as per Schneider et al. (11)] afforded recoveries of less than 2% for either isomer of DCPO. These results indicate a potential for rapid loss of the epoxide during liver homogenization and extraction. The rate of DCPO degradation was therefore measured in control blood and liver homogenate from both F344 rats and B6C3F1 mice. As shown in Table 1, the degradation of the DCPO isomers was quite rapid in both species. The half-life of the DCPO isomers in blood ranged from 1.04 to 2.42 min. The degradation of these analytes was also rapid in 10-fold-diluted liver homogenate, with halflives of less than 3.0 min for both isomers. Approximately 10-fold longer half-lives were observed in 100-fold dilutions of the liver homogenate, suggesting that the majority of in vitro degradation of DCPO isomers in the liver is enzyme-mediated. Heat-inactivated liver homogenate and buffer alone afforded half-lives slightly longer than those for 100-fold dilutions of liver homogenate, showing some chemical degradation of DCPO. The enzyme-mediated degradation of trans-DCPO was approximately 30% faster than that of the corresponding cis isomer in both species, which is consistent with the isomer-selective degradation rates observed by Schneider et al. for this compound (11). On the basis of these in vitro results, the tissue extraction technique was therefore modified to minimize the degradation of DCPO during sample preparation. Freshly isolated liver samples were immediately frozen in liquid nitrogen and crushed in a manual impact hammer, and the resulting granular sample was extracted directly with ethyl acetate. Recovery of DCPO isomers from liver samples fortified prior to freezing and extraction ranged from 10 to 41% (single extraction). Little additional DCPO was recovered with a second

Figure 1. Negative-ion chemical ionization spectra: (A) trans isomer of DCP (90 µg/mL) and (B) trans isomer of DCPO (50 µg/mL).

extraction of the liver samples (0-3%). These results indicate that the enzymatic degradation of DCPO is slowed by rapid freezing of the tissue sample. All subsequent in vivo experiments were therefore performed with this frozen tissue extraction method. Recoveries of DCPO isomers from rat and mouse whole blood were relatively high, ranging from 79 to 99%. The analysis of DCPO and DCP isomers was performed primarily via NCI-GC/MS analysis. Selected analyses for DCPO present in in vitro sample extracts were performed via PCI-GC/MS, as per the method of Schneider et al. (11). NCI-GC/MS was chosen for analysis of the in vivo samples as this method afforded detection limits of approximately 10 ng of DCP or DCPO per gram of blood or liver tissue, versus 2-fold higher detection limits via PCI-GC/MS. Deuterated analogues of both DCP and DCPO were employed in this assay to provide optimal correction for any potential loss of analyte(s) or systematic variability during sample extraction and analysis. The M - H- parent ion of DCP was monitored for the determination of DCP concentrations (m/z 109; Figure 1A). The M - D- parent ion for the 37Cl-labeled d4-DCP (m/z 114) was monitored for this internal standard, as the concentrations that were employed afforded a saturated GC/MS peak at m/z 112 during low-level analyses. The base peak observed for DCPO via NCI analysis was the Cl- fragment ion (Figure 1B). Due to the very low abundance of higher-mass ions for DCPO, quantitation of this metabolite and the deuterated internal standard was performed solely with the m/z 35 and 37 ions. Representative NCI chromatograms for the separation of the isomers of DCP are shown in Figure 2. Nearbaseline separation was achieved for the unlabeled and deuterated DCPO isotopomers with this assay (Figure 3). Similar separations of substantially deuterated compounds have been reported previously (15, 16). Confirma-

Metabolism of DCP in Rats and Mice

Figure 2. Selected ion monitoring NCI-GC/MS chromatograms of (A) isomers of cis- and trans-DCP in blood from a B6C3F1 mouse given 700 mg of DCP/kg ip (3.4 µg of cis-DCP/g, 7.0 µg of trans-DCP/g, and 12.4 µg of cis/trans-d4-DCP/g) and (B) the extract of control mouse blood (containing 12.4 µg of cis/transd4-DCP/g).

tion of DCPO detected in this assay was achieved by verifying that the m/z 35/37 ion ratio for the metabolites was within 30% of the ion ratio obtained from standard solutions of DCPO. No interference was observed for the isomers of DCP and DCPO in extracts of control blood (Figures 2B and 3C). The formation of DCPO as a metabolite of DCP was recently reported by Schneider et al. (11). In that study, the time course of DCPO formation in the liver tissue of male SW mice was followed after ip administration of a high dose of 350 or 700 mg of DCP/kg in a DMSO vehicle. Those dose levels were as high as the reported LD50 for this compound of 215-640 mg/kg (12). For comparative purposes, the in vivo experiment of Schneider et al. was repeated in the current study with the strain of mouse employed by Schneider (male SW), as well as male B6C3F1 mice, the strain generally employed in toxicity/oncogenicity studies with DCP (Figure 4). DCP and DCPO isomer concentrations in the liver of both strains of mice, following an ip dose of 700 mg/kg in a DMSO vehicle, were found to be comparable to those reported previously (11). Concentrations of these four compounds were also found to be comparable between the liver tissue and blood in both strains of mice. Levels of the trans isomer of DCP were found to be higher than those of the cis isomer in both liver and blood, which is consistent with the results of Schneider et al. In contrast, concentrations of cisDCPO were approximately 2-3 times that of the trans isomer, as was reported previously (11). These results indicate an isomer-specific formation and/or degradation of the epoxide metabolite of DCP. However, DCP at this high dose level was found to be acutely toxic and/or lethal in the mouse. Within a few minutes of administration, all mice in this experiment

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Figure 3. Selected ion monitoring NCI-GC/MS chromatograms of (A) a standard solution containing cis- and trans-DCPO each at 140 ng/mL, (B) isomers of cis- and trans-DCPO in blood from a B6C3F1 mouse given 100 mg of DCP/kg ip (0.5 ng of cis-DCPO/ g, 17.2 ng of trans-DCPO/g, and 58.5 ng of cis/trans-d4-DCPO/ g), and (C) the extract of control mouse blood (containing 58.5 ng of cis/trans-d4-DCPO/g).

displayed decreased movement. This progressed to shallow breathing and slight tremors in some animals within 10-20 min. The two 90 min time point SW mice died 60 and 76 min post-dosing. The two 90 min time point B6C3F1 mice were both moribund and therefore sacrificed at 48 and 51 min. Pairs of both strains of mice were treated with DMSO vehicle only, to obtain appropriate control tissue samples for this analysis. Administration of the DMSO alone to either strain of mouse was found to cause only slight sedation of the test animals. The fact that acute toxicity and/or lethality was observed at this extremely high dose level indicates that the normal physiology of these mice was significantly compromised. Subsequent experiments were therefore performed to investigate DCPO formation at sublethal doses of DCP. As shown in Figure 4, substantially lower levels of parent DCP isomers were found in the liver and blood of B6C3F1 mice receiving 100 mg of DCP/kg ip than in those given 700 mg of DCP/kg. The blood AUCs of cis- and transDCP were 90- and 32-fold lower, respectively, in the 100 mg/kg dose group than in the 700 mg/kg dose group (Table 2). These data indicate a nonlinear systematic bioavailability of parent DCP between the 100 and 700 mg/kg dose levels. This nonlinearity is presumably due to saturation of conjugative and hydrolytic pathways and is consistent with the dose-dependent depletion of glutathione, reported previously for DCP (12). No DCPO was seen in the livers of B6C3F1 mice given 100 mg of DCP/kg via ip administration (LOD ) 10 ng/ g; Figure 4). Low (17 ng/g) to nondetectable levels of DCPO were only found in the blood samples from this dose group. As seen with the parent compound, the rate of formation of DCPO was not linear between the dose

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Figure 4. Concentrations of DCP and DCPO isomers in the liver and blood of rats and mice treated with DCP.

levels of 100 and 700 mg/kg, ip. The AUC of cis- and trans-DCPO in blood of mice given 100 mg of DCP/kg was 203- and 62-fold lower, respectively, than DCPO levels in the high-dose group animals. This nonlinearity in the

rate of DCPO formation would correlate with saturation of DCP conjugation with glutathione at the 700 mg/kg dose level, affording more parent compound that is available for epoxidation.

Metabolism of DCP in Rats and Mice

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Table 2. In Vivo AUC Blood Values for Cis and Trans Isomers of DCP and DCPO blood AUC (0-60 min) values (µg min-1 g-1)a

a

species

dose level (mg/kg)

route

cis-DCP

trans-DCP

cis-DCPO

trans-DCPO

F344 rat B6C3F1 mouse B6C3F1 mouse B6C3F1 mouse SW mouse

100 100 100 700 700

po po ip ip ip

0.74 0.29b 44.3 3969 2908

4.50 0.92 181 5712 4615

0.29b 0.29b 0.42 85.4 33.0

0.29b 0.29b 0.43 26.8 15.8

n ) 2-4 animals per dose group. b All values ) ND (AUC calculated with values set to one-half of the detection limit).

The potential for epoxidation of DCP following the more relevant oral route was also examined. B6C3F1 mice were given 100 mg of DCP/kg via oral gavage, with corn oil as the vehicle. DCP concentrations in mouse liver were variable, but approximately equal to levels of parent compound following 100 mg/kg ip administration (Figure 4 and Table 2). DCP isomer concentrations in the blood of the B6C3F1 mouse, however, were approximately 5% of those seen via ip administration at this dose level. These data indicate a lower systematic availability of DCP via the oral route. Potential species differences in the fate of DCP epoxidation were examined in B6C3F1 mice and F344 rats, following oral administration of 100 mg/kg. The blood AUCs for the DCP isomers were approximately 3-5-fold higher in rats than in mice at this dose level. As was seen in all of the ip dose mouse groups, and has been reported previously for rats (17), blood levels of trans-DCP were 3-6-fold higher than those of the cis isomer in both rats and mice at this dose level. These data, suggesting a higher rate of metabolism for cis-DCP than for the trans isomer in both species, are consistent with previous studies showing the cis isomer as a better substrate for glutathione transferase than trans-DCP (12). No DCPO was seen in the liver or blood of B6C3F1 mice or F344 rats given 100 mg of DCP/kg via oral administration despite the bolus nature of the administration (Figure 4). The estimated AUC for cis- and trans-DCPO in these animals, based on half of the method detection limit, is 1% or less of the corresponding AUCs for this metabolite in B6C3F1 mice given 700 mg of DCP/kg via the ip route. These data demonstrate the efficiency and high capacity of the nonepoxidation metabolism of DCP in rodents. Serum levels of both ALT and AST were measured in each of the four treated mouse groups. Both enzymes were found to be elevated above controls in B6C3F1 mice given 100 or 700 mg/kg ip, within 60 min of administration (p < 0.05; Figure 5). No measurements after 20 min were available for the SW mice (700 mg/kg) due to lethality seen within 76 min post-dosing. No significant changes in either ALT or AST serum levels were seen in the B6C3F1 mice given 100 mg of DCP/kg via the oral route. These data indicate that DCP causes acute hepatic toxicity at dose levels of g100 mg/kg, when given via the ip route. No hepatoxicity was observed at 100 mg/kg following oral administration. These data are consistent the previous work of Miyaoka et al. (18), who found increases in the levels of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT), following oral administration of 300 mg of DCP/ kg to SPF-ICR male mice. They also reported no increase in the levels of liver enzymes at the lower dose of 100 mg/kg (po). Pretreatment with piperonyl butoxide was shown to lower the DCP-related increase in plasma GOT

Figure 5. Liver enzyme levels of B6C3F1 and Swiss-Webster mice treated with DCP: (A) serum ALT and (B) serum AST.

and GPT levels, which the authors attribute to suppression of a cytochrome P450-mediated toxic metabolite of DCP. In summary, significant nonlinearity was observed in the epoxidation of DCP in B6C3F1 mice between dose levels of 100 and 700 mg/kg (ip). Approximately 130-fold lower levels of DCPO formation were found at the 7-fold lower dose level, following ip administration. No detectable DCPO was found in rats or mice given the relatively high oral dose of 100 mg of DCP/kg. While DCPO may be a transient metabolite that rearranges to one or more ultimate mutagens, tissue levels of this compound should be a useful indicator of the relative rates of metabolism of DCP via epoxidation. The results of serum enzyme analysis indicate acute hepatic injury occurs only at dose levels of >100 mg of DCP/kg, following oral administration. From these data, we conclude that epoxidation of DCP to DCPO, in the rodent, is a very minor route of metabolism at dose levels that cause no acute hepatic injury or death.

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Acknowledgment. We thank Christine Thornton for her excellent technical assistance with the in vivo and in vitro studies. We also thank Doug Pearson for his preparation of DCPO and d4-DCPO.

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