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The effect of inducers and inhibitors of urethane metabolism on its in vitro and in vivo metabolism in rats
i f
CANCER LETTERS Cancer Letters 87 (1994) 145-150
The effect of inducers and inhibitors of urethane metabolism on its in vitro and in vivo metabolism in rats Gary P. Carlson Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907-47907. USA
Received 9 September 1994; accepted 23 September 1994
Abstract The activation of urethane (ethyl carbamate) is important in its exerting its carcinogenic effect. Rats were treated with inducers and inhibitors of urethane metabolism, and the conversion of [carbonyl-‘4C]urethane to 14C0, in vivo was measured. The cytochrome P-450 inducers, phenobarbital and &naphthoflavone, and esterase inhibitor, paraoxon, were without effect while the CYP2El inhibitor, diethyldithiocarbamate, decreased metabolism to about 3% of control. Ethanol administered acutely inhibited urethane metabolism. Pyridine, shown previously to enhance this metabolism in microsomal preparations, greatly inhibited it in vivo. The discordant results between the in vitro and in vivo studies may be related to the presence of pyridine acting as an inhibitor in whole animals and suggest that caution is needed in extrapolating from in vitro results to in vivo implications. Keywords:
Urethane;
Ethyl carbamate;
Carcinogen
metabolism;
1. Introduction The carcinogenicity of urethane has been recognized in laboratory animals for many years with liver and lung tumors being observed in mice and liver tumors in rats [2,18,19,23]. Urethane exerts its carcinogenic effect following bioactivation to vinyl carbamate and then to vinyl carbamate epoxide which forms RNA and DNA adducts and initiates tumorigenesis [3,16]. Urethane is also metabolized by esterases to yield ethanol, carbon dioxide and ammonia. The two step oxidation of urethane to the active vinyl carbamate epoxide is catalysed primarily by 0304-3835/94/%07.00 0 1994 Elsevier Science Ireland SSDI 0304-3835(94)03590-F
Pyridine;
Ethanol
CYP2El (71. A number of compounds have been found to be good inducers of this isozyme ineluding ethanol which can increase the metabolism of urethane or decrease it depending upon the conditions of exposure [14,25,26]. Pyridine is also an excellent inducer of CYP2El apoprotein and associated activities in both the liver [12,13] and lung [I]. We recently studied the effect of pyridine on the microsomal metabolism of urethane to CO, and binding in vivo of urethane to macromolecules [21]. When rats and mice were treated with 200 mg/kg pyridine i.p. 18 h prior to sacrifice, a dosing regimen known to cause induction of CYP2El and
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146
G.P. Carlson/Cancer
attendant activities, it increased the in vitro metabolism of [carbonyl-14C]urethane to CO2 in hepatic microsomal preparations although there was little effect on pulmonary microsomes. In contrast, when the animals were treated with pyridine and 18 h later were administered [ethyl- 14C]urethane, pyridine greatly diminished the binding of the ethyl group to DNA, RNA and protein in both tissues in both species. The purpose of the present experiments was to examine further the discordant results observed between these two experiments by treating rats with pyridine and examining the in vivo metabolism of [carbonyl- 14C]urethane to 14C02 and the in vitro binding of [ethyl-‘4C]urethane to macromolecules. Additional inducers and inhibitors of cytochrome P-450 and esterases were also studied for comparative purposes. 2. Materials and methods 2. I. Animals Adult male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were allowed rodent laboratory chow no. 5001 (Purina Mills, Inc., St. Louis, MO) and water ad libitum. Lights were on a 12-h 1ight:dark cycle. 2.2. Chemicals Diethyldithiocarbamate, urethane, P-naphthoflavone, calf thymus DNA, NADPH, and paraoxon were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium phenobarbital and pyridine were from J.T. Baker, Inc. (Phillipsburg, NJ). All other chemicals were of reagent grade quality or better. [carbonyl-i4C]Urethane (specific activity 12.7 mCi/mmol, radiochemical purity 1 98%) and [ethyl-‘4C]urethane (specific activity 6.85 mCi/ mmol, radioactivity 1 98%) were purchased from Sigma Chemical Co. Hi-Ionic Fluor liquid scintillation cocktail was obtained from Parkard Instruments (Meriden, CT). 2.3. In vivo studies Rats were treated with inducers and inhibitors. For the induction schedules, phenobarbital was given at a dose of 80 mglkglday i.p. for 4 days and fi-naphthoflavone (in corn oil) at a dose of 40
Lett. 87 (1994) 145-150
mglkglday i.p. for 3 days. Urethane was given 24 h after the last dose. Pyridine was given at a dose of 200 mg/kg i.p. one or 18 h prior to the urethane. Ethanol was administered either acutely (4 ml ethanol/kg, given by gavage as a 25% solution, one h before the urethane) or over a period of 3 weeks as a 10% solution in the drinking water with removal of the ethanol 24 h before the urethane. The inhibitors diethyldithiocarbamate (400 mg/kg i.p.) and paraoxon (0.4 mg/kg i.p.) were administered 30 min before urethane. [carbonyl- 14C]Urethane was administered at a dose of 2.5 mg/kg and 1.0 @i per animal i.p. Using a modification of a previously described apparatus [22] individual rats were placed in all glass Roth-Delmar metabolism cages. Air was passed through the chamber at a rate of 500-650 ml/min under slightly negative pressure and through three gas washing bottles, the first of which contained ethanol to collect any unmetabolized urethane. Two one-ml samples were taken for scintillation counting 1,2,4 and 6 h during exposure. A second trap contained a mixture of ethanolamine and 2butoxyethanol (1:2) to trap 14C02. The sampling was the same. The data are presented as the cumulative percentage of the administered dose. A third trap, similar to the second, was used to detect any breakthrough. Since this turned out to be very small (usually less than one-half percent of the administered dose in 6 h), the amounts of 14C02 measured in the third trap are not reported. The 14C-urethane and 14C02 were determined by counting 1 ml aliquots of the traps in 15 ml of Packard Hi-Ionic Fluor liquid scintillation cocktail using a Beckman model 1801 liquid scintillation counter. 2.4. In vitro studies To determine the effect of pyridine on the binding of the ethyl group from urethane to DNA and protein, [ethyl-‘4C]urethane (0.5 mM, 1 PCi) was incubated with 10 mM MgCl*, 1 mM NADPH, 1.0 PM paraoxon (to inhibit esterase metabolism of the urethane) and whole homogenate equivalent to 100 mg tissue in 0.1 M potassium phosphate buffer (pH 7.4) in a final volume of 1.2 ml. Incubations were carried out at 37°C for 30 min in parafilm covered Corex tubes in a Dubnoff metabolic shaker.
G.P. Carlson / Cancer Let!. 87 (1994)
Binding to DNA and protein was determined using the method of Fossa et al. [6] with the macromolecules being extracted and purified by the Kirby procedure as modified by Diamond et al. [5]. To the incubation mixture were added 2 ml of an aqueous solution of 1% NaCl, 1% triisopropylnaphthalene sulfonic acid, 6% see-butyl alcohol and 6% p-aminosalicylate (20 ml/g tissue). One ml of chlorofommsoamyl alcohol (24:l) was added, and the samples were placed in 30-ml Corex tubes and shaken for 10 min followed by centrifugation at 10 000 g for 30 min. Protein separated at the interface of the two layers. The top aqueous layer containing the nucleic acids was extracted again with chlorofotmisoamyl alcohol. DNA was precipitated by the addition of one volume of 2-ethoxyethanol and removed by centrifuging at 10 000 rpm for 10 min. The DNA was washed 5 times with 1.0 ml of ethanol and three times with 1.0 ml of diethyl ether. After drying with nitrogen, it was solubilized in 2 ml of 0.1 N NaOH. The purity of the DNA was determined from the A260/A280 ratio and the amount from the absorbance at 260 nm. The protein was washed in a similar fashion. After solubilizing in 0.1 N NaOH, a portion was used for quantification using the Lowry procedure [ 171. 14C was measured using Packard Hi-Ionic Fluor liquid scintillation cocktail and a Beckman model 3801 liquid scintillation counter. In some cases rats were treated with 200 mg/kg pyridine 18 h before sacrifice. In other studies the pyridine was added to the incubation mixture at a final concentration of 1 mM. In other experiments calf thymus DNA (4.29 mg/incubation) was added to the incubation mixture. 2.5. Statistical analysis Means and standard errors were calculated for all groups. Comparisons among groups were made using an ANOVA followed by a Student-NeumanKeul’s test with the level of significance chosen as P c 0.05. For the sake of clarity, only differences from control are indicated in the Tables. 3. Results When [carbonyl-14C]urethane was administered to control rats i.p., a little more than one-half of
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147
it was eliminated as 14C02 in 6 h (Table 1). A high dose of paraoxon was used to inhibit the esterase. Even though the animals demonstrated signs of toxicity, there was no effect on the metabolism of urethane. However, when diethyldithiocarbamate (DDTC), a relatively specific inhibitor of CYP2El [8,15] was given to the animals, there was a very great decrease in urethane metabolism to a level of about 3% that of the controls indicating the importance of this isozyme to the metabolism of urethane in vivo. The classic inducers of CYP2B and CYPlA isozymes, /3-naphthoflavone and phenobarbital, were without effect. When pyridine was administered acutely, i.e., one h before the urethane, it greatly inhibited urethane metabolism to a level near that observed with the inhibitor DDTC (Table 1). Nearly as great a decrease was observed when the pyridine was administered a full 18 h prior to the urethane. In a parallel experiment, ethanol administered at a high dose of 4 ml/kg one h before the urethane also greatly diminished the metabolism of urethane. When rats were administered 10% ethanol in the drinking water for a period of 3 weeks but were given water for the last 24 h so that blood levels would have returned to control level, there was no diminution in urethane metabolizing activity. Urethane 14C-labeled in the ethyl position was used to examine the effect of pyridine on the binding in vitro of the ethyl moiety to DNA and protein in the livers of rats. Small amounts did bind to both macromolecules (Table 2). Only in one study did pyridine appear to have any influence on the binding. In general when the pyridine was either administered to the animals or added in vitro it had little effect even when additional calf thymus DNA was added. 4. Discussion Urethane is like many small molecular weight carcinogens in that its bioactivation to an active form is associated with CYP2El [8]. It would appear logical that an increase in the activity of this isozyme could be associated with an enhanced biotransformation and hence toxicity such as that observed with carbon tetrachloride [4, lo]. One excellent inducer of this isozyme is pyridine [ 1,12,13] which potentiates the hepatotoxicity and pneumo-
G.P. Carlson/Cancer
148 Table I Influence
of inducers
Treatment
and inhibitors Number animals
on the metabolism of
Trap
Leff. 87 119941 145-150
of [carbon_$‘4C]urethane
lb
Carbon
dioxideC
Ih Control Paraoxond DDTCe Phenobarbital’ @-Naphthoflavones Pyridine (acute) h Pyridine’ Ethanol (acute)j Ethanol (3 weeks)l
10 4 4 4 4 4 4 4 6
0.52 3.36 0.48 0.68 2.13 0.06 2.44 0.41 1.19
zt 0.08 + 2.36 f 0.18 ZL0.05 zt 1.92 zt 0.04 zt 0.96 f 0.15 + 0.15
12.5 9.8 I.1 12.6 10.3 0.4 1.3 0.6 9.9
to CO1 in vivoa
2h f 0.6 zt 0.5 l 0.1* + 2.5 l 1.7 l 0.2* f 0.3* f 0.2* f 1.2
26.8 22.4 1.4 29.5 23.7 0.6 2.0 1.1 22.2
4h + 1.4 zt I.1 l 0.2* f 1.9 + 1.8 + 0.2* f 0.3; l 0.3’ f 1.F
44.9 39.5 1.6 49.6 48.0 1.2 2.9 2.2 41.0
6h zt f + zt + f
1.8 2.2 0.2’ 1.6 4.0 0.1* l 0.5; f 0.5* i 2.1
52.1 48.8 1.6 59.3 56.9 1.6 3.8 3.5 51.9
f zt f f zt + f
2.2 2.1 0.2* 2.0 2.4 0.1’ 0.8; l 0.7* f 2.1
aRats were given various treatments and then administered 2.5 mg/kg [carbony/-ethyl-‘4C]carbamate i.p. *rap I contained 10% ethanol to trap exhaled urethane. Value is percentage of administered dose at end of experiment. CTrap contained ethanolamine:2-butoxyethanol (1:2). Value is cumulative percentage of administered dose. Asterisk indicates value is significantly different from control (P < 0.05). dParaoxon was given at a dose of 0.4 mgikg i.p. 30 min before the urethane. eDiethyIdithiocarbamate was given at a dose of 400 mg/kg i.p. 30 min before the urethane. ‘Phenobarbital was given at a dose of 80 mg/kg/day i.p. for 4 days. Rats were given urethane 24 h after last dose. so-Naphthoflavone was given at a dose of 40 mg/kg/day i.p. for 3 days. Rats were given the urethane 24 h after last dose. hPyridine was given at a dose of 200 mg/kg i.p. one h before the urethane. ‘Pyridine was given at a dose of 200 mg/kg i.p. 18 h before the urethane. iEthanol was given at a dose of 4 ml/kg by gavage one h before the urethane. kEthanol was administered as a 10% solution in the drinking water for 3 weeks. Rats were given water instead of ethanol for the last 24 h before the urethane.
Table 2 Binding of [erhyl-‘4C]urethane protein in vitro
to
rat
hepatic
DNA
Treatment
N
DNAa
Proteinb
Control Pyridinec
4 4
Control PyridineC Controld Pyridine c.4
4 4 4 4
Control Pyridinee
4 4
40.1 25.7 10.8 10.7 19.9 21.4 23.4 17.3
5.92 7.39 1.29 1.43 NDs ND 3.51 3.92
apmols bpmols
14C bound/mg 14C bound/mg
f f + f
1.5 2.4’ 1.5 0.7 l 2.2 f 5.3 + 1.8 f 3.5
DNA. protein.
c200 mg/kg i.p. 18 h prior to measurements. d4.29 mg calf thymus DNA added per incubation. eAdded in vitro at final concentration of 1 mM. fSigniticantly different from control, P < 0.05. gNot determined.
and
zt 0.41 ZIZ2.89 zt 0.05 f 0.15
f 0.27 + 0.27
toxicity of carbon tetrachloride [4]. However, when the interaction between pyridine and urethane was examined in a previous study, the conclusions drawn from in vitro measurements differed from those from in vivo measurements [21]. Specifically, the administration of pyridine (200 mg/kg i.p. 18 h prior to sacrifice of the rats) greatly increased the microsomal metabolism of urethane to COZ, but when urethane binding was determined in whole animals, there was significant protection against its binding to macromolecules. In the present experiments, treatments with various inducers and inhibitors were compared for their effects on urethane metabolism to CO1 in vivo. Surprisingly, paraoxon had no effect despite the fact that it is a good inhibitor of urethane metabolism in vitro [21]. On the other hand DDTC was an extremely effective inhibitor demonstrating the predominant role of CYP2El. This was substantiated by the finding that /3naphthoflavone and phenobarbital, inducers of CYPlA and CYP2B isozymes, respectively, did
G. P. Carlson / Cancer Lett. 87 (1994)
not alter urethane metabolism and thus are not considered to be involved, at least at the low dose of urethane used in these particular studies. When ethanol was administered acutely, it inhibited the metabolism of urethane which is in agreement with the results of other workers [14,25,26]. This suggests that the ethanol is likely acting as a competitive substrate, an effect not observed when ethanol was administered for 3 weeks but withdrawn for 24 h to allow for its clearance from the tissues prior to the urethane administration. While pyridine is a good inducer of CYP2El and as such increases the metabolism of urethane measured using microsomes [21], just as it enhances the metabolism of a number of other substrates such as nitrosodimethylamine [ 1,111, aniline [ 111, p-nitrophenol [ 1,121, 2-butanol [20], l,l-dichloro-2,2,2-trifluoroethane [24], halothane [24] and 1,1,2,2-tetrafluoro1-(2,2,2_trifluoroethoxy)-ethane [9] when microsomal preparations are used, in the whole animal pyridine either one or 18 h prior to the urethane, inhibits its metabolism. This is not surprising since pyridine is a substrate for CYP2El [12] although it is not entirely clear -whether it is acting as a competitive substrate or is acting noncompetitively as suggested by Kaul and Novak [l 11. In either event, the inhibition is reversible as demonstrated by the fact that the induction of the metabolism of urethane to CO2 is observed in microsomal preparations [2 l] presumably as a result of the washing out of pyridine and/or its metabolites. The results of these studies suggest that caution is needed in extrapolating from in vitro results to in vivo implications. Acknowledgements This study was supported in part by NIH Grant ES04362 and an Indiana Elks grant through the Purdue University Cancer Center. The author is very appreciative of the able technical assistance of Feng Jiang. References [l]
Carlson, G.P. and Day, B.J. (1992) Induction by pyridine of cytochrome P-450IIEI and xenobiotic metabolism in rat lung and liver. Pharmacology, 44, 117-123.
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PI Dahl, G.A., Miller, E.C. and Miller, J.A. (1980) Comparative carcinogenicities and mutagenicities of vinyl carbamate, ethyl carbamate and ethyl N-hydroxycarbamate. Cancer Res., 40, 1194-1203. [31 Dahl, G.A., Miller, J.A. and Miller, E.C. (1978) Vinyl carbamate as a promutagen and a more carcinogenic analog of ethyl carbamate. Cancer Res., 38, 3793-3804. D.B. (1993) [41 Day, B.J., Carlson, G.P. and DeNicola, Potentiation of carbon tetrachloride-induced hepatotoxicity and pneumotoxicity by pyridine. J. Biochem. Toxicol., 8, 11-18. 151 Diamond, L., Defendi, V. and Brookes, P. (1967) The interaction of 7,12-dimethylbenz(a)anthracene with cells sensitive and resistant to toxicity caused by this carcinogen. Cancer Res., 27, 890-897. 161 Fossa, A.A., Baird, W.M. and Carlson, G.P. (1985) Distribution of urethane and its binding to DNA, RNA and protein in SENCAR and BALB/c mice following oral and dermal administration. J. Toxicol. Environ. Health, 15, 635-654. 171 Guengerich, F.P. and Kim, D.-H. (1991) Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of l,N6-ethenoadenosine. Chem. Res. Toxicol., 4, 413-421. F.P., Kim, D-H. and Iwasaki, M. (1991) [81 Guengerich, Role of human cytochrome P-450 HE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol., 4, 168-179. R. and Dekant, W. 191 Herbst, J., Koster, U., Kerssebaum, (1994) Role of P-4502EI in the metabolism of 1,1,2,2tetrafluoro-1-(2,2,2_trifluoroethoxy)-ethane. Xenobiotica, 24, 507-516. M. (1985) Carbon 1101 Johansson, I. and Ingelman-Sundberg, tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450. FEBS Lett., 183, 265-269. Ill1 Kaul, K.L. and Novak, R.F. (1987) Inhibition and induction of rabbit liver microsomal cytochrome P-450 by pyridine. J. Pharmacol. Exp. Ther., 243, 384-390. 1121 Kim, S.G. and Novak, R.F. (1990) Induction of rat hepatic P-45011El (CUP 2El) by pyridine: Evidence for a role of protein synthesis in the absence of transcriptional activation. Biochem. Biophys. Res. Commun., 166, 1072- 1079. 1131 Kim, S.G., Williams, D.E., Schuetz, E.G., Guzelian, P.S. and Novak, R.F. (1988) Pyridine induction of cytochrome P-450 in the rat: role of P-450j (alcoholinducible form) in pyridine N-oxidation. J. Pharmacol. Exp. Ther., 246, 1175-I 182. I141 Kurata, N., Hurst, H.E., Kemper, R.A. and Waddell, W.J. (1991) Studies on induction of metabolism of ethyl carbamate in mice by ethanol. Drug Metab. Dispos., 19, 239-240. I151 Lauriault, V.V., Khan, S. and O’Brien, P.J. (1992) Hepatocyte cytotoxicity induced by various hepatotoxins mediated by cytochrome P-450,,,,: protection with diethyldithiocarbamate administration. Chem.-Biol. Interact., 81, 271-289.
G.P. Carlson/CancerLat. 87 (1994) 145-150
150
1161Leithauser,
M.T., Liem, A., Stewart, B.C., Miller, E.C. and Miller, J.A. (1990) I,@-Ethenoadenosine formation, mutagenicity and murine tumor induction as indicators of the generation of an electrophilic epoxide metabolite of the closely related carcinogens ethyl carba-
mate (urethane) and vinyl carbamate. Carcinogenesis, 11, 463-473. N.J., Farr, A.L. and Randall, 1171 Lowry, O.H., Rosebrough, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. iI81 Mirvish, S.S. (1968) The carcinogenic action and metabolism of urethan and N-hydroxyurethan. Adv. Cancer Res., 11, I-42. I191 Nettleship, A., Henshaw, P. and Meyer H. (1943) Induction of pulmonary tumors in mice with ethyl carbamate (urethan). J. Nat]. Cancer Inst., 4, 309-319. WI Page, D.A. and Carlson, G.P. (1993) Effect of pyridine on the hepatic and pulmonary metabolism of 2-butanol in rat and rabbit. J. Toxicol. Environ. Health, 38. 369-379. PI Page, D.A. and Carbon, G.P. (1994) The effect of pyridine on the in vitro and in vivo metabolism of ethyl car-
P21
~231
[241
[251
P61
bamate (urethane) by rat and mouse. Carcinogenesis, in press. Paustenbach, D.J., Carlson, G.P., Christian, J.E. and Born, G.S. (1986) A comparative study of the pharmacokinetics of carbon tetrachloride in the rat following repeated inhalation exposures of 8 and 11.5 h/day. Fundam. Appl. Toxicol., 6, 484-497. Schmahl, D., Port, R. and Wahrendorf, J. (1977) A doseresponse study on urethane carcinogenesis in rats and mice. Int. J. Cancer, 19, 77-80. Urban, G., Speerschneider, P. and Dekant. W. (1994) Metabolism of chlorofluorocarbon substitute l,ldichloro-2,2,2_trifluoroethane by rat and human liver microsomes: the role of cytochrome P-450 2El. Chem. Res. Toxicol., 7, 170-176. Yamamoto, T., Pierce, W.M., Hurst, H.E.. Chen, D. and Waddell, W.J. (1988) Inhibition of the metabolism of urethane by ethanol. Drug Metab. Dispos., 16. 355-358. Yamamoto. T., Pierce, W.M.. Hurst, H.E.. Chen, D. and Waddell. W.J. (1990) Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems. Drug Metab. Dispos.. 18. 276-280.
CANCER LETTERS Cancer Letters 87 (1994) 145-150
The effect of inducers and inhibitors of urethane metabolism on its in vitro and in vivo metabolism in rats Gary P. Carlson Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907-47907. USA
Received 9 September 1994; accepted 23 September 1994
Abstract The activation of urethane (ethyl carbamate) is important in its exerting its carcinogenic effect. Rats were treated with inducers and inhibitors of urethane metabolism, and the conversion of [carbonyl-‘4C]urethane to 14C0, in vivo was measured. The cytochrome P-450 inducers, phenobarbital and &naphthoflavone, and esterase inhibitor, paraoxon, were without effect while the CYP2El inhibitor, diethyldithiocarbamate, decreased metabolism to about 3% of control. Ethanol administered acutely inhibited urethane metabolism. Pyridine, shown previously to enhance this metabolism in microsomal preparations, greatly inhibited it in vivo. The discordant results between the in vitro and in vivo studies may be related to the presence of pyridine acting as an inhibitor in whole animals and suggest that caution is needed in extrapolating from in vitro results to in vivo implications. Keywords:
Urethane;
Ethyl carbamate;
Carcinogen
metabolism;
1. Introduction The carcinogenicity of urethane has been recognized in laboratory animals for many years with liver and lung tumors being observed in mice and liver tumors in rats [2,18,19,23]. Urethane exerts its carcinogenic effect following bioactivation to vinyl carbamate and then to vinyl carbamate epoxide which forms RNA and DNA adducts and initiates tumorigenesis [3,16]. Urethane is also metabolized by esterases to yield ethanol, carbon dioxide and ammonia. The two step oxidation of urethane to the active vinyl carbamate epoxide is catalysed primarily by 0304-3835/94/%07.00 0 1994 Elsevier Science Ireland SSDI 0304-3835(94)03590-F
Pyridine;
Ethanol
CYP2El (71. A number of compounds have been found to be good inducers of this isozyme ineluding ethanol which can increase the metabolism of urethane or decrease it depending upon the conditions of exposure [14,25,26]. Pyridine is also an excellent inducer of CYP2El apoprotein and associated activities in both the liver [12,13] and lung [I]. We recently studied the effect of pyridine on the microsomal metabolism of urethane to CO, and binding in vivo of urethane to macromolecules [21]. When rats and mice were treated with 200 mg/kg pyridine i.p. 18 h prior to sacrifice, a dosing regimen known to cause induction of CYP2El and
Ltd. All rights reserved
146
G.P. Carlson/Cancer
attendant activities, it increased the in vitro metabolism of [carbonyl-14C]urethane to CO2 in hepatic microsomal preparations although there was little effect on pulmonary microsomes. In contrast, when the animals were treated with pyridine and 18 h later were administered [ethyl- 14C]urethane, pyridine greatly diminished the binding of the ethyl group to DNA, RNA and protein in both tissues in both species. The purpose of the present experiments was to examine further the discordant results observed between these two experiments by treating rats with pyridine and examining the in vivo metabolism of [carbonyl- 14C]urethane to 14C02 and the in vitro binding of [ethyl-‘4C]urethane to macromolecules. Additional inducers and inhibitors of cytochrome P-450 and esterases were also studied for comparative purposes. 2. Materials and methods 2. I. Animals Adult male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were allowed rodent laboratory chow no. 5001 (Purina Mills, Inc., St. Louis, MO) and water ad libitum. Lights were on a 12-h 1ight:dark cycle. 2.2. Chemicals Diethyldithiocarbamate, urethane, P-naphthoflavone, calf thymus DNA, NADPH, and paraoxon were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium phenobarbital and pyridine were from J.T. Baker, Inc. (Phillipsburg, NJ). All other chemicals were of reagent grade quality or better. [carbonyl-i4C]Urethane (specific activity 12.7 mCi/mmol, radiochemical purity 1 98%) and [ethyl-‘4C]urethane (specific activity 6.85 mCi/ mmol, radioactivity 1 98%) were purchased from Sigma Chemical Co. Hi-Ionic Fluor liquid scintillation cocktail was obtained from Parkard Instruments (Meriden, CT). 2.3. In vivo studies Rats were treated with inducers and inhibitors. For the induction schedules, phenobarbital was given at a dose of 80 mglkglday i.p. for 4 days and fi-naphthoflavone (in corn oil) at a dose of 40
Lett. 87 (1994) 145-150
mglkglday i.p. for 3 days. Urethane was given 24 h after the last dose. Pyridine was given at a dose of 200 mg/kg i.p. one or 18 h prior to the urethane. Ethanol was administered either acutely (4 ml ethanol/kg, given by gavage as a 25% solution, one h before the urethane) or over a period of 3 weeks as a 10% solution in the drinking water with removal of the ethanol 24 h before the urethane. The inhibitors diethyldithiocarbamate (400 mg/kg i.p.) and paraoxon (0.4 mg/kg i.p.) were administered 30 min before urethane. [carbonyl- 14C]Urethane was administered at a dose of 2.5 mg/kg and 1.0 @i per animal i.p. Using a modification of a previously described apparatus [22] individual rats were placed in all glass Roth-Delmar metabolism cages. Air was passed through the chamber at a rate of 500-650 ml/min under slightly negative pressure and through three gas washing bottles, the first of which contained ethanol to collect any unmetabolized urethane. Two one-ml samples were taken for scintillation counting 1,2,4 and 6 h during exposure. A second trap contained a mixture of ethanolamine and 2butoxyethanol (1:2) to trap 14C02. The sampling was the same. The data are presented as the cumulative percentage of the administered dose. A third trap, similar to the second, was used to detect any breakthrough. Since this turned out to be very small (usually less than one-half percent of the administered dose in 6 h), the amounts of 14C02 measured in the third trap are not reported. The 14C-urethane and 14C02 were determined by counting 1 ml aliquots of the traps in 15 ml of Packard Hi-Ionic Fluor liquid scintillation cocktail using a Beckman model 1801 liquid scintillation counter. 2.4. In vitro studies To determine the effect of pyridine on the binding of the ethyl group from urethane to DNA and protein, [ethyl-‘4C]urethane (0.5 mM, 1 PCi) was incubated with 10 mM MgCl*, 1 mM NADPH, 1.0 PM paraoxon (to inhibit esterase metabolism of the urethane) and whole homogenate equivalent to 100 mg tissue in 0.1 M potassium phosphate buffer (pH 7.4) in a final volume of 1.2 ml. Incubations were carried out at 37°C for 30 min in parafilm covered Corex tubes in a Dubnoff metabolic shaker.
G.P. Carlson / Cancer Let!. 87 (1994)
Binding to DNA and protein was determined using the method of Fossa et al. [6] with the macromolecules being extracted and purified by the Kirby procedure as modified by Diamond et al. [5]. To the incubation mixture were added 2 ml of an aqueous solution of 1% NaCl, 1% triisopropylnaphthalene sulfonic acid, 6% see-butyl alcohol and 6% p-aminosalicylate (20 ml/g tissue). One ml of chlorofommsoamyl alcohol (24:l) was added, and the samples were placed in 30-ml Corex tubes and shaken for 10 min followed by centrifugation at 10 000 g for 30 min. Protein separated at the interface of the two layers. The top aqueous layer containing the nucleic acids was extracted again with chlorofotmisoamyl alcohol. DNA was precipitated by the addition of one volume of 2-ethoxyethanol and removed by centrifuging at 10 000 rpm for 10 min. The DNA was washed 5 times with 1.0 ml of ethanol and three times with 1.0 ml of diethyl ether. After drying with nitrogen, it was solubilized in 2 ml of 0.1 N NaOH. The purity of the DNA was determined from the A260/A280 ratio and the amount from the absorbance at 260 nm. The protein was washed in a similar fashion. After solubilizing in 0.1 N NaOH, a portion was used for quantification using the Lowry procedure [ 171. 14C was measured using Packard Hi-Ionic Fluor liquid scintillation cocktail and a Beckman model 3801 liquid scintillation counter. In some cases rats were treated with 200 mg/kg pyridine 18 h before sacrifice. In other studies the pyridine was added to the incubation mixture at a final concentration of 1 mM. In other experiments calf thymus DNA (4.29 mg/incubation) was added to the incubation mixture. 2.5. Statistical analysis Means and standard errors were calculated for all groups. Comparisons among groups were made using an ANOVA followed by a Student-NeumanKeul’s test with the level of significance chosen as P c 0.05. For the sake of clarity, only differences from control are indicated in the Tables. 3. Results When [carbonyl-14C]urethane was administered to control rats i.p., a little more than one-half of
145-150
147
it was eliminated as 14C02 in 6 h (Table 1). A high dose of paraoxon was used to inhibit the esterase. Even though the animals demonstrated signs of toxicity, there was no effect on the metabolism of urethane. However, when diethyldithiocarbamate (DDTC), a relatively specific inhibitor of CYP2El [8,15] was given to the animals, there was a very great decrease in urethane metabolism to a level of about 3% that of the controls indicating the importance of this isozyme to the metabolism of urethane in vivo. The classic inducers of CYP2B and CYPlA isozymes, /3-naphthoflavone and phenobarbital, were without effect. When pyridine was administered acutely, i.e., one h before the urethane, it greatly inhibited urethane metabolism to a level near that observed with the inhibitor DDTC (Table 1). Nearly as great a decrease was observed when the pyridine was administered a full 18 h prior to the urethane. In a parallel experiment, ethanol administered at a high dose of 4 ml/kg one h before the urethane also greatly diminished the metabolism of urethane. When rats were administered 10% ethanol in the drinking water for a period of 3 weeks but were given water for the last 24 h so that blood levels would have returned to control level, there was no diminution in urethane metabolizing activity. Urethane 14C-labeled in the ethyl position was used to examine the effect of pyridine on the binding in vitro of the ethyl moiety to DNA and protein in the livers of rats. Small amounts did bind to both macromolecules (Table 2). Only in one study did pyridine appear to have any influence on the binding. In general when the pyridine was either administered to the animals or added in vitro it had little effect even when additional calf thymus DNA was added. 4. Discussion Urethane is like many small molecular weight carcinogens in that its bioactivation to an active form is associated with CYP2El [8]. It would appear logical that an increase in the activity of this isozyme could be associated with an enhanced biotransformation and hence toxicity such as that observed with carbon tetrachloride [4, lo]. One excellent inducer of this isozyme is pyridine [ 1,12,13] which potentiates the hepatotoxicity and pneumo-
G.P. Carlson/Cancer
148 Table I Influence
of inducers
Treatment
and inhibitors Number animals
on the metabolism of
Trap
Leff. 87 119941 145-150
of [carbon_$‘4C]urethane
lb
Carbon
dioxideC
Ih Control Paraoxond DDTCe Phenobarbital’ @-Naphthoflavones Pyridine (acute) h Pyridine’ Ethanol (acute)j Ethanol (3 weeks)l
10 4 4 4 4 4 4 4 6
0.52 3.36 0.48 0.68 2.13 0.06 2.44 0.41 1.19
zt 0.08 + 2.36 f 0.18 ZL0.05 zt 1.92 zt 0.04 zt 0.96 f 0.15 + 0.15
12.5 9.8 I.1 12.6 10.3 0.4 1.3 0.6 9.9
to CO1 in vivoa
2h f 0.6 zt 0.5 l 0.1* + 2.5 l 1.7 l 0.2* f 0.3* f 0.2* f 1.2
26.8 22.4 1.4 29.5 23.7 0.6 2.0 1.1 22.2
4h + 1.4 zt I.1 l 0.2* f 1.9 + 1.8 + 0.2* f 0.3; l 0.3’ f 1.F
44.9 39.5 1.6 49.6 48.0 1.2 2.9 2.2 41.0
6h zt f + zt + f
1.8 2.2 0.2’ 1.6 4.0 0.1* l 0.5; f 0.5* i 2.1
52.1 48.8 1.6 59.3 56.9 1.6 3.8 3.5 51.9
f zt f f zt + f
2.2 2.1 0.2* 2.0 2.4 0.1’ 0.8; l 0.7* f 2.1
aRats were given various treatments and then administered 2.5 mg/kg [carbony/-ethyl-‘4C]carbamate i.p. *rap I contained 10% ethanol to trap exhaled urethane. Value is percentage of administered dose at end of experiment. CTrap contained ethanolamine:2-butoxyethanol (1:2). Value is cumulative percentage of administered dose. Asterisk indicates value is significantly different from control (P < 0.05). dParaoxon was given at a dose of 0.4 mgikg i.p. 30 min before the urethane. eDiethyIdithiocarbamate was given at a dose of 400 mg/kg i.p. 30 min before the urethane. ‘Phenobarbital was given at a dose of 80 mg/kg/day i.p. for 4 days. Rats were given urethane 24 h after last dose. so-Naphthoflavone was given at a dose of 40 mg/kg/day i.p. for 3 days. Rats were given the urethane 24 h after last dose. hPyridine was given at a dose of 200 mg/kg i.p. one h before the urethane. ‘Pyridine was given at a dose of 200 mg/kg i.p. 18 h before the urethane. iEthanol was given at a dose of 4 ml/kg by gavage one h before the urethane. kEthanol was administered as a 10% solution in the drinking water for 3 weeks. Rats were given water instead of ethanol for the last 24 h before the urethane.
Table 2 Binding of [erhyl-‘4C]urethane protein in vitro
to
rat
hepatic
DNA
Treatment
N
DNAa
Proteinb
Control Pyridinec
4 4
Control PyridineC Controld Pyridine c.4
4 4 4 4
Control Pyridinee
4 4
40.1 25.7 10.8 10.7 19.9 21.4 23.4 17.3
5.92 7.39 1.29 1.43 NDs ND 3.51 3.92
apmols bpmols
14C bound/mg 14C bound/mg
f f + f
1.5 2.4’ 1.5 0.7 l 2.2 f 5.3 + 1.8 f 3.5
DNA. protein.
c200 mg/kg i.p. 18 h prior to measurements. d4.29 mg calf thymus DNA added per incubation. eAdded in vitro at final concentration of 1 mM. fSigniticantly different from control, P < 0.05. gNot determined.
and
zt 0.41 ZIZ2.89 zt 0.05 f 0.15
f 0.27 + 0.27
toxicity of carbon tetrachloride [4]. However, when the interaction between pyridine and urethane was examined in a previous study, the conclusions drawn from in vitro measurements differed from those from in vivo measurements [21]. Specifically, the administration of pyridine (200 mg/kg i.p. 18 h prior to sacrifice of the rats) greatly increased the microsomal metabolism of urethane to COZ, but when urethane binding was determined in whole animals, there was significant protection against its binding to macromolecules. In the present experiments, treatments with various inducers and inhibitors were compared for their effects on urethane metabolism to CO1 in vivo. Surprisingly, paraoxon had no effect despite the fact that it is a good inhibitor of urethane metabolism in vitro [21]. On the other hand DDTC was an extremely effective inhibitor demonstrating the predominant role of CYP2El. This was substantiated by the finding that /3naphthoflavone and phenobarbital, inducers of CYPlA and CYP2B isozymes, respectively, did
G. P. Carlson / Cancer Lett. 87 (1994)
not alter urethane metabolism and thus are not considered to be involved, at least at the low dose of urethane used in these particular studies. When ethanol was administered acutely, it inhibited the metabolism of urethane which is in agreement with the results of other workers [14,25,26]. This suggests that the ethanol is likely acting as a competitive substrate, an effect not observed when ethanol was administered for 3 weeks but withdrawn for 24 h to allow for its clearance from the tissues prior to the urethane administration. While pyridine is a good inducer of CYP2El and as such increases the metabolism of urethane measured using microsomes [21], just as it enhances the metabolism of a number of other substrates such as nitrosodimethylamine [ 1,111, aniline [ 111, p-nitrophenol [ 1,121, 2-butanol [20], l,l-dichloro-2,2,2-trifluoroethane [24], halothane [24] and 1,1,2,2-tetrafluoro1-(2,2,2_trifluoroethoxy)-ethane [9] when microsomal preparations are used, in the whole animal pyridine either one or 18 h prior to the urethane, inhibits its metabolism. This is not surprising since pyridine is a substrate for CYP2El [12] although it is not entirely clear -whether it is acting as a competitive substrate or is acting noncompetitively as suggested by Kaul and Novak [l 11. In either event, the inhibition is reversible as demonstrated by the fact that the induction of the metabolism of urethane to CO2 is observed in microsomal preparations [2 l] presumably as a result of the washing out of pyridine and/or its metabolites. The results of these studies suggest that caution is needed in extrapolating from in vitro results to in vivo implications. Acknowledgements This study was supported in part by NIH Grant ES04362 and an Indiana Elks grant through the Purdue University Cancer Center. The author is very appreciative of the able technical assistance of Feng Jiang. References [l]
Carlson, G.P. and Day, B.J. (1992) Induction by pyridine of cytochrome P-450IIEI and xenobiotic metabolism in rat lung and liver. Pharmacology, 44, 117-123.
145-150
149
PI Dahl, G.A., Miller, E.C. and Miller, J.A. (1980) Comparative carcinogenicities and mutagenicities of vinyl carbamate, ethyl carbamate and ethyl N-hydroxycarbamate. Cancer Res., 40, 1194-1203. [31 Dahl, G.A., Miller, J.A. and Miller, E.C. (1978) Vinyl carbamate as a promutagen and a more carcinogenic analog of ethyl carbamate. Cancer Res., 38, 3793-3804. D.B. (1993) [41 Day, B.J., Carlson, G.P. and DeNicola, Potentiation of carbon tetrachloride-induced hepatotoxicity and pneumotoxicity by pyridine. J. Biochem. Toxicol., 8, 11-18. 151 Diamond, L., Defendi, V. and Brookes, P. (1967) The interaction of 7,12-dimethylbenz(a)anthracene with cells sensitive and resistant to toxicity caused by this carcinogen. Cancer Res., 27, 890-897. 161 Fossa, A.A., Baird, W.M. and Carlson, G.P. (1985) Distribution of urethane and its binding to DNA, RNA and protein in SENCAR and BALB/c mice following oral and dermal administration. J. Toxicol. Environ. Health, 15, 635-654. 171 Guengerich, F.P. and Kim, D.-H. (1991) Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of l,N6-ethenoadenosine. Chem. Res. Toxicol., 4, 413-421. F.P., Kim, D-H. and Iwasaki, M. (1991) [81 Guengerich, Role of human cytochrome P-450 HE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol., 4, 168-179. R. and Dekant, W. 191 Herbst, J., Koster, U., Kerssebaum, (1994) Role of P-4502EI in the metabolism of 1,1,2,2tetrafluoro-1-(2,2,2_trifluoroethoxy)-ethane. Xenobiotica, 24, 507-516. M. (1985) Carbon 1101 Johansson, I. and Ingelman-Sundberg, tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450. FEBS Lett., 183, 265-269. Ill1 Kaul, K.L. and Novak, R.F. (1987) Inhibition and induction of rabbit liver microsomal cytochrome P-450 by pyridine. J. Pharmacol. Exp. Ther., 243, 384-390. 1121 Kim, S.G. and Novak, R.F. (1990) Induction of rat hepatic P-45011El (CUP 2El) by pyridine: Evidence for a role of protein synthesis in the absence of transcriptional activation. Biochem. Biophys. Res. Commun., 166, 1072- 1079. 1131 Kim, S.G., Williams, D.E., Schuetz, E.G., Guzelian, P.S. and Novak, R.F. (1988) Pyridine induction of cytochrome P-450 in the rat: role of P-450j (alcoholinducible form) in pyridine N-oxidation. J. Pharmacol. Exp. Ther., 246, 1175-I 182. I141 Kurata, N., Hurst, H.E., Kemper, R.A. and Waddell, W.J. (1991) Studies on induction of metabolism of ethyl carbamate in mice by ethanol. Drug Metab. Dispos., 19, 239-240. I151 Lauriault, V.V., Khan, S. and O’Brien, P.J. (1992) Hepatocyte cytotoxicity induced by various hepatotoxins mediated by cytochrome P-450,,,,: protection with diethyldithiocarbamate administration. Chem.-Biol. Interact., 81, 271-289.
G.P. Carlson/CancerLat. 87 (1994) 145-150
150
1161Leithauser,
M.T., Liem, A., Stewart, B.C., Miller, E.C. and Miller, J.A. (1990) I,@-Ethenoadenosine formation, mutagenicity and murine tumor induction as indicators of the generation of an electrophilic epoxide metabolite of the closely related carcinogens ethyl carba-
mate (urethane) and vinyl carbamate. Carcinogenesis, 11, 463-473. N.J., Farr, A.L. and Randall, 1171 Lowry, O.H., Rosebrough, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. iI81 Mirvish, S.S. (1968) The carcinogenic action and metabolism of urethan and N-hydroxyurethan. Adv. Cancer Res., 11, I-42. I191 Nettleship, A., Henshaw, P. and Meyer H. (1943) Induction of pulmonary tumors in mice with ethyl carbamate (urethan). J. Nat]. Cancer Inst., 4, 309-319. WI Page, D.A. and Carlson, G.P. (1993) Effect of pyridine on the hepatic and pulmonary metabolism of 2-butanol in rat and rabbit. J. Toxicol. Environ. Health, 38. 369-379. PI Page, D.A. and Carbon, G.P. (1994) The effect of pyridine on the in vitro and in vivo metabolism of ethyl car-
P21
~231
[241
[251
P61
bamate (urethane) by rat and mouse. Carcinogenesis, in press. Paustenbach, D.J., Carlson, G.P., Christian, J.E. and Born, G.S. (1986) A comparative study of the pharmacokinetics of carbon tetrachloride in the rat following repeated inhalation exposures of 8 and 11.5 h/day. Fundam. Appl. Toxicol., 6, 484-497. Schmahl, D., Port, R. and Wahrendorf, J. (1977) A doseresponse study on urethane carcinogenesis in rats and mice. Int. J. Cancer, 19, 77-80. Urban, G., Speerschneider, P. and Dekant. W. (1994) Metabolism of chlorofluorocarbon substitute l,ldichloro-2,2,2_trifluoroethane by rat and human liver microsomes: the role of cytochrome P-450 2El. Chem. Res. Toxicol., 7, 170-176. Yamamoto, T., Pierce, W.M., Hurst, H.E.. Chen, D. and Waddell, W.J. (1988) Inhibition of the metabolism of urethane by ethanol. Drug Metab. Dispos., 16. 355-358. Yamamoto. T., Pierce, W.M.. Hurst, H.E.. Chen, D. and Waddell. W.J. (1990) Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems. Drug Metab. Dispos.. 18. 276-280.