Chem. Res. Toxicol. 1995,8, 987-992
987
Species-Dependent Differences in Biotransformation Pathways of 2-Methylpropene (Isobutene) Miranda Cornet,*>+,$ Andre Callaerts,? Ute Jorritsma,g$"Hermann Bolt,g Antoine Vercruysse,t and Vera Rogiers? Department of Toxicology, Vrzje Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium, and Institut fur Arbeitsphysiologie an der Universitat Dortmund, Ardeystrasse 67, 0-44139 Dortmund 1, Germany Received February 21, 1995@
The biotransformation of 2-methylpropene, a gaseous alkene widely used in industry, was investigated in vitro in liver tissue of rats, mice, and humans. Interspecies comparison revealed that the lowest levels of the primary epoxide metabolite were detected in incubations of 2-methylpropene with human liver homogenate, followed by rat and mouse, respectively. Among the human liver samples, however, important interindividual variations were observed. Out of the 16 samples analyzed, only 2 contained measurable epoxide amounts, while in the other samples only traces were detectable. The involvement of rat liver cytochrome P450 2E1 in the activation of 2-methylpropene to its epoxide 2-methyl-1,2-epoxypropanehas been established. The lower capacity of the mixed function oxidase system in human liver samples compared to rodents is confirmed. Concerning epoxide detoxifylng enzymes, a high microsomal epoxide hydrolase activity was observed in human liver tissue and a n intermediate in rat liver, while a low activity was measured in mouse liver. These findings were inversely correlated with the epoxide levels measured in vitro in liver tissue of the three species studied. It can be concluded that, a s far as the in vitro metabolism of 2-methylpropene is concerned, neither mouse nor rat represents a good model for the human situation. Although, the same biotransformation pathways are involved, marked quantitative differences in epoxide levels were observed. The results indicate that human liver tissue is exposed in vitro to smaller concentrations of the primary metabolite 2-methyl-172-epoxypropane than rodent liver.
Introduction 2-Methylpropene (MP)l or isobutene is a gaseous alkene widely used as a monomer in the industrial production of adhesives, plastics, butyl rubber, and other polymers ( I ) . Occupational exposure to MP can thus occur during production, storage, and manufacture of the chemical in an industrial environment. Despite the risk for human exposure, the metabolic fate and toxicity of MP are still unknown in humans. In rodents, some data are available. 2-Methyl-1,2-epoxypropane(MEP) has been identified as the primary metabolite of MP in liver tissue of rodents. MP is converted, in vitro (2) as well as in vivo (3),to this reactive epoxide intermediate. It can subsequently be hydrolyzed to a diol or conjugated to glutathione to form more hydrophilic metabolites (2), as shown in Figure 1. Due to their electrophilic reactivity, epoxides may react with nucleophilic centers in the cell, inducing alterations of critical cellular macromol-
2-methylpropene (MP)
P4502E1
C-CH, CH:
+
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+
p b
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Toxicity
2-methyl- 1.2-epoxypropane
(MEPI
/\
Epoxide hydrolase
Glutathione S-transferase
CH l 3 OH I
CH3-C-CH, OH
* Address correspondence to this author at UCB S.A., Pharmaceutical Division. R&D. Deuartment of Toxicoloeical Research. Chemin du Foriest, B-1420 Braiie-l'Alleud, Belgium.-Tel: 32.2.386.21.39; Fax: 32.2.386.27.98. Vrije Universiteit Brussel. 7 Present address: UCB S.A., Pharmaceutical Division, R&D, Department of Toxicological Research, Chemin du Foriest, B-1420 Braine-l'Alleud, Belgium. 5 Universitat Dortmund. 'I Present address: Institut fur Pharmakologie und Toxikologie, Georg-August-Universitat Gottingen, Robert-Koch-Strasse 40, D-37075 Gottingen, Germany. Abstract published in Advance ACS Abstracts, August 15, 1995. Abbreviations: AH, aniline 4-hydroxylase; CDNB, 1-chloro-2,4dinitrobenzene; ECOD, 7-ethoxycoumarin 0-deethylase; EH, epoxide hydrolase; GST, glutathione S-transferase;MAb, monoclonal antibody; MEP, 2-methyl-l.2-epoxypropane;MP, 2-methylpropene; P450, cytochrome P450.
Binding t o RNA, DNA, macromolecules
CH3,
2-methyl.l,2-propanediol
CH OH
la I
CH3-C-CH2
CH
SG
IS I
or CH3-C-CH2
SG
OH
glutathione conjugate
Figure 1. Biotransformation scheme of MP.
ecules, such as DNA, RNA, and proteins, which may disturb normal biochemical processes and may lead to cytotoxic and genotoxic effects (4). The latter has been confirmed for MEP, which, in contrast to the parent compound MP, displays a mutagenic effect at high concentrations in Salmonella typhimurium strains TA100, TA1535, and TA102 (5)and in the in vitro micronucleus test using human lymphocytes (6). For 1,3-butadiene, an alkene closely related to MP, important differences in toxicokinetic parameters have been observed between
0893-228x/95/2708-0987$09.00/0 0 1995 American Chemical Society
988 Chem. Res. Toxicol., Vol. 8, No. 7, 1995
Cornet e t al.
Table 1. Sample Characteristics, MEP Levels, Enzymatic Activities, and P450 Content of 16 Human Livers" human liver 1 2 3 4 5 6 7
8 9 10 11 12 13 14 15 16
sex
age (years)
cause of death
MEP
P450
ECOD
AH
mEH
cEH
GST
F
64 25 56 27 22 43 55 64 66 44 64 50 28 40 64 70
cerebral trauma suicide cerebral anoxia road accident (cerebral trauma) road accident (cerebral trauma) cerebral trauma suicide brain hemorrhage brain hemorrhage suicide brain hemorrhage cerebral anoxia cerebral trauma brain hemorrhage brain hemorrhage brain hemorrhage
trace 219 trace trace trace trace trace trace 156 trace trace trace trace trace trace trace
0.845 0.309 0.477 0.528 0.438 0.521 0.466 0.670 0.271 0.441 0.802 0.431 0.280 0.827 0.693 0.538
0.515 0.760 0.357 0.258 0.375 0.437 0.445 0.446 0.513 0.476 0.580 0.308 0.581 0.600 0.438 0.393
0.278 0.558 0.370 0.122 0.276 0.376 0.411 0.361 0.561 0.484 0.351 0.412 0.594 0.538 0.558 0.510
9.030 8.082 8.852 8.796 9.546 12.260 16.251 15.769 12.753 14.322 16.062 14.178 15.967 16.393 16.275 15.924
0.255 0.355 0.166 0.247 0.146 0.244 0.259 0.282 0.256 0.254 0.266 0.212 0.220 0.242 0.113 0.113
1.524 1.876 1.946 2.048 1.621 1.029 2.370 3.335 1.641 2.914 2.705 2.254 1.217 1.758 2.205 2.016
M M M
M M F M
F M
F F M F F M
a M = male, F = female. MEP level is expressed as pmol/(mg of total protein.20 min); P450 content is expressed as nmob'mg of microsomal protein; ECOD activity is expressed as nmol of 7-hydroxycoumarin/(mg of microsomal proteinemin); AH activity is expressed as nmol of p-aminophenoll(mg of microsomal proteinmin); microsomal EH activity is expressed as nmol of diol/(mg of microsomal proteimmin); cytosolic EH activity is expressed as nmol of diol/(mg of cytosolic proteimmin); GST activity is expressed as unitdmg of cytosolic protein.
rodent species (7). Mice achieve higher levels of reactive epoxide metabolites (butadiene monoxide and diepoxide) (8,9>,probably contributing to the higher susceptibility of this species to carcinogenic effects of inhaled 1,3butadiene (10). Hence, the aim of the present study was to investigate the biotransformation of MP in vitro in liver tissue of different species, including man. Through this approach an interspecies comparison can be made and the difficulty of extrapolating animal data to man can be evaluated (11, 12). Extrapolation of in vitro results to the in vivo situation, however, still remains necessary (13). Since variations in enzymatic activities controlling epoxide formation and detoxification can significantly contribute to species differences in MP metabolism, the activities of cytochrome P450 (P450)-dependentmonooxygenases, epoxide hydrolase (EH)and glutathione S-transferase (GST), were measured for the three species studied. The involvement of specific P450 enzymes in the activation of MP to the reactive MEP metabolite was studied in rat liver tissue by means of an immunoinhibition assay.
Materials and Methods Chemicals. Glucose &phosphate, NADP+, and l-chloro-2,4dinitrobenzene (CDNB) were obtained from Sigma Chemical Co. (St. Louis, MO). Glucose-6-phosphate dehydrogenase and reduced glutathione were purchased from Boehringer (Mannheim, Germany). MP was from Messer Griesheim (Dusseldorf, Germany). Nembutal was obtained from Sanofi-Ceva (Brussels, Belgium), while heparin (5000 IU/mL) came from Novo Industries (Brussels, Belgium). 7-Ethoxycoumarin was purchased from Janssen Chimica (Geel, Belgium), 7-hydroxycoumarin from Ega Chemie (Steinheim, Germany), and aniline sulfate from Fluka (Bornem, Belgium). MEP was synthesized by Prof. M. Vandewalle (Department of Organic Chemistry, Rijksuniversiteit Gent, Belgium). [3H]trans-Stilbene oxide and [ 3 H l ~ i s stilbene oxide were kindly provided by Dr. J. Meijer (Department of Cell Research, Swedish University of Agricultural Sciences, Uppsala, Sweden). All other chemicals were readily available commercial products. Liver Samples. Male Sprague-Dawley rats (200-300 g) were obtained from Iffa Credo (Brussels, Belgium). Male mice (25-35 g) bred from C57 black females and CBA males (Animalarium W B , Brussels, Belgium) were used. The animals had free access to food and drinking water. Human liver samples were obtained from organ donors (details are given in Table 1). Only tissue not used for transplantation was involved.
Liver tissue was obtained shortly after circulatory arrest from renal transplant donors. Preparation of Subcellular Fractions. 9OOOg supernatant, microsomes, and cytosol from rodent liver were prepared as described previously ( 1 4 ) . Human liver samples were divided into small cubes immediately after isolation and were stored in liquid nitrogen until use. Further handling of human samples was the same a s for rodent liver. Determination of the MEP Concentration. The incubations were carried out in gas-tight headspace vials containing a NADPH-generating system (2.65 mM glucose 6-phosphate, 0.26 mM NADP-, and 2.3 U/mL glucose-6-phosphate dehydrogenase), 3.31 mM MgC12, 34 mM sodium phosphate buffer (pH 7.61, and 9OOOg supernatant (5-15 mg of protein) in a final volume of 1.51 mL. After a preincubation period of 10 min at 37 "C, the reaction was started by injection of MP gas (final concentration 10 000 ppm) through the Teflon-coated septum of the vials. The reaction was carried out a t 37 "C under gentle shaking of the vials. At appropriate reaction times, 1.5 mL samples of the gas phase were taken through the septum and were injected into the gas sample loop of the gas chromatograph. The gas chromatographic conditions were as described previously ( 2 ) . The total amount of MEP present in gas and liquid phase was calculated ( 2 ) . Determination of the P450 Content. The microsomal P450 content was determined from the CO difference spectrum, as described by Omura and Sat0 (15). Contamination of human liver samples with cytochrome P420 was minimal (generally 5 15%) and consequently does not influence the P450 results. Determination of Enzymatic Activities. All enzymatic assays were performed under conditions where the amount of product formed was linearly proportional to the amount of protein and to the incubation time.
(A) 7-Ethoxycoumarin 0-Deethylase (ECOD) Activity. Microsomal ECOD activity was determined according to Paterson et al. (16). The incubation mixture contained 0.5 mM NADP+, 5 mM MgC12, 5 mM glucose 6-phosphate, 0.6 mg of albumin, 1U of glucose-6-phosphate dehydrogenase, microsomes (0.15 mg of protein), and 10.8 mM potassium phosphate buffer (pH 7.4) in a total volume of 1 mL. After a preincubation period of 2 min a t 37 "C, the reaction was started by addition of 7-ethoxycoumarin (final concentration 0.5 mM). The reaction was stopped after 20 min by addition of 200 ,uL of 10% w/v trichloroacetic acid. The 7-hydroxycoumarin formed was extracted and measured fluorimetrically (16). (B)Aniline 4-Hydroxylase(AH) Activity. Microsomal AH activity was measured a s described by Imai et al. (17). The incubation mixture contained 5.4 mM nicotinamide, 0.13 mM NADP', 12.5 mM MgC12, 3.12 mM glucose 6-phosphate, 0.6 mg of albumin, 2 U/mL glucose-6-phosphate dehydrogenase, mi-
Chem. Res. Toxicol., Vol. 8, No. 7, 1995 989
Biotransformation P a t h w a y s of 2-Methylpropene crosomal suspension (0.50-0.75 mg of protein), and 50 mM Tris buffer (pH 7.4) in a final volume of 0.5 mL. The reaction was started by addition of aniline sulfate (final concentration 2 mM). The assay was carried out for 20 min at 37 "C and was terminated by the addition of 250 p L of 10% w/v trichloroacetic acid. After centrifugation, 350 p L of the supernatant was added to 150 p L of 2 M NazC03 and 250 pL of 1% w/v phenol in 2% w/v NaOH and incubated for 30 min at 37 "C. The resulting color was measured a t 620 nm. (C) GST Activity. Cytosolic GST activity was measured toward the general substrate CDNB according t o the spectrophotometrical method of Habig e t al. (18). The incubation mixture contained cytosol (0.50-0.75 mg of protein), 1.6 mM glutathione, 1.6 mM CDNB, and 160 mM sodium phosphate buffer (pH 6.5) in a final volume of 1.25 mL. The formation of glutathione conjugate was measured at 340 nm. (D) EH Activities. Cytosolic and microsomal E H activities were assayed using the method of Gill et al. (19), modified according to Moody e t al. (20). [3Hltrans- and [3Hlcis-stilbene oxide were used a s substrates for the cytosolic and the microsomal forms of the enzyme, respectively. For the determination of the cytosolic EH activity, the incubation mixture contained cytosol (0.25 mg of protein), 0.5 mM diethyl maleate, and 64 mM sodium phosphate buffer (pH 7.6) in a final volume of 199 pL. The reaction was started by addition of 1pL of [3Hltransstilbene oxide (final concentration 25 pM). For the microsomal E H activity the reaction mixture contained microsomes (2540 pg of protein) and 0.1 M glycine buffer (pH 9.0) in a final volume of 199 ,uL. The reaction was started by addition of 1pL of [3H]cis-stilbene oxide (final concentration 25 ,uM). For both assays, the reaction was terminated after 10 min by addition of 200 ,uL of dodecane, and the diol formed was measured radiometrically (19, 20). Determination of Protein Concentration, Protein concentrations were determined according to the procedure of Bradford (21),using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Miinchen, Germany) with bovine serum albumin as a standard. Immunoinhibition Study. Inhibiting monoclonal antibodies (MAb), raised in mice against specific rat P450 enzymes, were kindly provided by Prof. Gelboin (Laboratory of Molecular Carcinogenesis, National Institutes of Health, Bethesda, MD). Anti-P4501Al MAb (clone 1-7-1)which cross reacts with P4501A2 (22),anti-P4502B1 MAb (clone 2-66-3 and clone 4-29-5) which cross reacts with P4502B2 (23),anti-P4502Cll MAb (clone 1-6811) which cross reacts with P4502C6 (241, and anti-P4502E1 MAb (clone 1-91-3) (25) were used. An additional rat antiP4502B1 (which may cross react with P4502B21, raised in mice, was a gift of Prof. Beaune (Department of Biochemistry, CHU Necker-Inserm U75, Paris, France). After reaction of the MAb (MAb amounts producing maximal inhibition were used) with microsomes (ca. 2 mg of protein) for 20 min at room temperature in 67 mM sodium phosphate buffer (pH 7.6), the NADPH-generating system (2.65 mM glucose 6-phosphate, 0.26 mM NADP+, and 2.3 U/mL glucose-6phosphate dehydrogenase) and 3.31 mM MgClz were added (in a total volume of 1.51 mL), and the reaction vials were preincubated for 10 min at 37 "C. The assay was started by addition of 10 000 ppm MP. After a reaction time of 15 min, the MEP concentration was measured a s described previously (2). Statistical Analysis. The statistical analysis was achieved by t-tests using the Software Package of the Social Sciences (SPSS/PC+ V2.0) (26).
Results Epoxide Steady State Concentrations. MEP concentrations, measured in liver 9OOOg supernatant of the three species studied, are given at different time points in Figure 2. It appears that the highest epoxide level was found in liver tissue of mouse, followed by rat and man. About half the amount of MEP present in mouse
;1 se:i
4000
Human
3200
i
5m
2400
t 4
1600
;
800
0
6
9
12
15
20
30
40
50
60
Time (mi")
Figure 2. MEP concentration in liver tissue of different species a s a function of time. Results are presented a s mean & SD [n = 6 for r a t and mouse, n = 2 for man (human livers 2 and 91, 14 other h u m a n samples contained only trace amounts].
.z
1.5
1.5
1.2
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0.9
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0.6
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0.3
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0.0
a
Mouse
Rat
3
5
Human
Figure 3. P450 content and ECOD and AH activity in liver tissue of different species. Results are presented as mean f SD (n = 6 for r a t and mouse, n = 16 for man). P450 content is expressed as nmol/mg of microsomal protein, ECOD activity is expressed a s nmol of hydroxycoumarin/(mg of microsomal proteiwmin), and AH activity is expressed a s nmol of p aminophenol/(mg of microsomal proteinsmin). (a) Significantly different from rat ( p ~ 0 . 0 5 )(b) . Significantly different from mouse and rat ( p
