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SESOBIOTIC.~ , 1988, VOL. 18, NO. 10, 1109-1118
Metabolism of diamantane by rat liver microsomal cytochromes P-450"
PETR HODEK, PAVEL JANSCAK, PAVEL ANZENBACHER-f, J IR i BURKHARDS, JOSEF JANKUJf, and LUDBK VODIcKAJf Department of Biochemistry, Charles University, Albertov 2030, 128 40 Praha 2, $Laboratory of Synthetic Fuels, Prague Institute of Chemical Technology, Suchbatarova 5, 166 28 Praha 6, Czechoslovakia
Received 20 October 1987; accepted 20 June 1988
1. Diamantane binds to liver microsomes from phenobarbital-treated rats with an apparent Ks value of 5.2 x lO-'mol/l. This value being lower than that obtained for perhydrophenanthrene indicates that diamantane is very strongly bound to microsomal cytochrome P-450.
2. Metabolic studies show that liver microsomes from phenobarbital-treated rats readily metabolize diamantane to mono-, di- and possibly tri-hydroxy derivatives, whereas liver microsomes from P-naphthoflavone-induced rats do not bind this hydrocarbon or metabolize it.
3. Reconstituted cytochromes P-450 b and e were more efficient in the hydroxylation of diamantane than liver microsomes; metabolites formed by the reconstituted system do not include all the products formed by microsomes, which indicates the involvement of forms of cytochrome P-450 other than the isozymes b and e.
Introduction Metabolism of non-polar substrates in liver is performed mainly by the mixed
function oxidase system with the cytochromes P-450 acting as the terminal oxidase (White and Coon 1980, Ortiz de Montellano 1986, Ruckpaul and Rein 1984). The two most studied families of rat liver microsomal cytochromes P-450 involve the phenobarbital (PB)-inducible cytochromes P-450b and e and the B-naphthoflavone (BNF)- or 3-methylcholanthrene-inducible cytochromes P-450c and d.
Recently, it has been shown that there are differences between the cytochromes P-450 and P-448 in the architecture of their binding sites. Cytochromes P-450 are able to bind bulky, non-planar molecules such as adamantane (White et al. 1984), whereas the cytochromes P-448 bind rather planar structures such as polycyclic aromatic hydrocarbons (Lewis et al. 1986).
The binding of substrates to cytochrome P-450 can be monitored by difference absorption spectrophotometry as this binding is associated with a shift of the Soret peak (Schenkman et al. 1981). The value of the apparent spectral dissociation constant Ki, calculated from analogy with the Michaelis-Menten equation for enzyme kinetics (Schenkman et al. 1967)) is taken as a measure of the binding affinity of a substrate. In most cases, this also reflects the ability of a substrate to be metabolized by the cytochrome P-450 system.
*Dedicated to Prof. Dr. Z. %pal, who inspired P-450 study in our laboratory. T o whom correspondence should be addressed
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1110 P. Hodek et al.
4
1
8
7
4 6
11
10
9
Figure 1. Structure of (A) adamantane and (€3) diamantane. Equivalent positions of diamantane: apical 4 and 9; medial 1,6,2,11,7,12; secondary 5,14,8,13,3,10.
In the present study, the binding of two structurally related substrates, adamantane and diamantane (figure 1) to liver microsomes, as well as to purified cytochromes P-450, was examined, and the products of their microsomal metabol- ism were identified. The main reason for studying the metabolism and binding of these compounds is their therapeutic use, since derivatives of adamantane are used in the treatment of Parkinsonism (Wesemann 1983).
Materials and methods Materials
Liver microsomes were prepared by differential centrifugation of liver homogenates from PB- or BNF-pretreated male Wistar rats (Van der Hoeven and Coon 1974). Rats (body wt 180-200g) were treated with 0.1% (w/v) solution of phenobarbital in drinking water for ten days. Another group of rats was injected intraperitoneally with a solution of 8-naphthoflavone (60 mg/kg) in three doses with an interval of two days; the last dose was applied one day before termination.
Cytochrome P-450b and e were isolated by a method based on hydrophobic chromatography on 1 -adamantanecarbonyl-aminohexyl-Sepharose 4B according to Anzenbacher et al. (1984). After ion- exchange chromatography on DEAE-Sephacel@ (Pharmacia, Uppsala, Sweden), cytochrome P-450e was obtained directly on washing the column, and its purification was completed by chromatography on hydroxyiapatite prepared in our laboratory according to Levin (19621, giving 19.0 nmol cytochrome/mg. The cytochrome P-450b (16.1 nmol cytochrome/mg) was obtained after further ion-exchange chroma- tography on DEAE-Sepharose CI, 6B (Pharmacia, Uppsala, Sweden), under conditions described by Anzenbacher et al. (1984). Chromatography on hydroxylapatite (Schenkman et al. 1982) was used in both cases for removal of detergents and concentration of the preparation. To decrease the effect of the degradation products of the nonionic detergent Renex 690 on cytochrome P-450 stability, all solutions contained 0.1 M mannitol. Both preparations were homogeneous electrophoretically.
NADPH-cytochrome P-450 reductase was obtained by the procedure described by Yasukochi et al. (1 979).
All chemicals used were of reagent grade purity. The adamantane was a product of Organopharma (Usti nad Labem, CSSR) and the diamantane was synthetized according to the method of Courtney et al. (1972); the purity of both substances was checked by g.1.c. (see below).
Assays The cytochrome P-450 contents was determined according to Omura and Sato (1964); protein
determination was based on the method of Lowry et al. (1951) with bovine serum albumin as standard.
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Metabolism of diamantane by P-450 1111
The amount of remaining detergent Renex 690 was checked according to the method of Goldstein and Blecher (1975); the detergent was removed by adsorption on silica gel (Anzenbacher et al. 1984). Reductase activity was determined by reduction of cytochrome c as described by Vermilion and Coon (1978).
Binding of substrates to cytochrome P-450 The binding of substrates to cytochrome P-450 as well all others spectral measurements were done
with a Specord M 4 0 spectrophotometer (Carl Zeiss, Jena, DDR). Microsomes were diluted to a protein concentration 1.13 mg/ml and divided into two cuvettes with 1 cm optical path. A solution of substrate in methanol, not exceeding 5% of the total volume, was added to the contents of the sample cuvette. The same volume of methanol was added to the contents of the reference cuvette to eliminate differences in concentration and effects of methanol.
Metabolism of adamantane and diamantane by a reconstituted cytochrome P-450 system and by microsomes Reconstitution was achieved by mixing 1 nmol purified cytochrome P-450 isozyme and NADPH-
cytochrome P-450 reductase in 1 : 1 molar ratio in presence of 70pg of dilauroylphosphatidykholine. After 3 minutes of preincubation at 25°C the other components of the reaction mixture were added (in amounts given for 1 ml): 0.1 pmol EDTA, 7.5 pmol MgCl,, 8.0 pmol glucose 6-phosphate, 0.8 pmol NADP', 1.0 IU glucose 6-phosphate dehydrogenase, 4000 IU catalase in 0.1 M K/PO, buffer pH 7.4. Substrates were added as methanolic solutions; final concentration being 0.3 mM (adamantane) and 0.13 mM (diamantane).
In the experiments with microsomes, all components added after preincubation were the same as for the reconstituted system; with microsomes in a final concentration of 2.8 mg/ml being used instead of cytochrome P-450 isozyme, the reductase and phospholipid.
Reaction was initiated by addition of NADP'. After incubation (3-6 h at 25"C), 1 ml of chloroform was added to stop the reaction. After 2min of vortexing, the chloroform layer was separated by centrifugation (1 5 min, SOOOg), removed and concentrated by evaporation. Extraction was done with each sample, and samples were prepared in triplicate. The reference sample (without substrate) was treated in the same way.
Influence of diamantane or adamantane on aminopyrine N-demethylation N-Demethylation in microsomes was studied almost under the same conditions as above, only the
temperature was higher (37°C) and catalase was omitted. Aminopyrine concentration was 6 mM in a final volume of 3.0ml. Adamantane and diamantane dissolved in dimethyl sulphoxide was added to the reaction mixture containing microsomes. The reaction rate was determined from the amount of formaldehyde produced per minute per nmol cytochrome P-450. For the blank, only the reaction mixture with dimethyl sulphoxide was used. Formaldehyde was determined according to Nash (1953).
Determination of metabolites Concentrated extracts were analysed by g.1.c. on Chromatone N-AW-DMCS (Lachema, Brno,
CSSR) with Silicone XF-1150 (Alltech, USA) using a Chrom 5 gas chromatograph (L.P., Prague, CSSR) with a flame ionization detection connected with a CI 100 computing integrator (L.P., Prague, (5SSR). The chromatography of adamantane and its mono-hydroxy derivatives was carried out at 110°C; the analysis of di-hydroxy derivatives was done at 150°C. Diamantane and mono-hydroxy diamantanes were analysed at 150"C, whereas di-hydroxy derivatives were chromatographed at 180°C. This two-temperature regime of analysis was used to get an appropriate resolution of analysed compounds. The standards of adamantane derivatives (ketone of adamantane; 1 - and 2-hydroxy adamantane; 1,4- and 1,3-dihydroxy adamantane) and diamantane derivates (ketone of diamantane; 1-, 3- and 4-hydroxy diamantane; 1,4-, 1,6-, 4,9- and 3,9-dihydroxy diamantane) were synthesized according to Geluk and Keizer (1972), Jankd et al. (1975, 1981 a, 1981 b). To obtain unequivocal identification of diamantane and adamantane metabolites, the mixture of respective standards was chromatographed immediately before the analysis of the samples. This arrangement was necessary to ensure a highly precise comparison of respective retention times. The retention times of the standards at conditions used are shown in figures 3 (A, B) and 4 by arrows.
Results Interaction of diamantane with cytochrome P-450
Binding of diamantane to PB-pretreated rat liver microsomes induces a typical Type I difference spectrum (maximum at 387 nm and minimum at 421 nm). Almost the same spectral feature is exhibited by two typical substrates, adamantane and perhydrophenanthrene which were used for comparison. The spectral data for diamantane (table 1) show that this substrate exhibits the lowest KL value. The same conclusion follows from the spectral studies with purified cytochrome P- 450b.
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Table I . Spectral data for binding of hydrocarbons to cytochrome P-450 in liver microsomes from phenobarbital-pretreated rats.
Substrate AA,,,/nmol P-450
(nmol-’)
Diamantane Adamantane Perhydrophenathrene
5.21 k0.43 x lo-’ 0.0233 &0.0004 1.3410.12 x 0.0133 _+0-0003 1.18*0.20 x 0.0269 & 0.0008
Mean values & S.D. calculated according to Wilkinson 1961.
1 1 I f
0 0.1 0.2 0.3 0.4
Concentration of diamantane or adamantane (mM)
Figure 2. Effects of adamantane and diamantane 011 the rate of aminopyrine N-demethylation in phenobarbital-treated rat liver microsomes.
Data is expressed as nmol formaldehyde/min per nmol of P-450, after addition of adamantane (0-0) or diamantane (0-0).
On the other hand, when diamantane or adamantane was added to the liver microsomes of BNF-pretreated rats, spectral changes of about twenty times lower values were detected. Due to very small spectral changes in this case, no KL and AA,,,/nmol cytochrome P-450 values could be obtained.
Influence of diamantane and adamantane on aminopyrine N-demethylation Both adamantane and diamantane are able to decrease significantly the N -
demethylation of aminopyrine in PB-pretreated rat liver microsomes (figure 2). On the other hand, no effect of these hydrocarbons on the aminopyrine N- demethylation was observed when microsomal fraction from BNF-pretreated rats was used.
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I l l 4 P. Hodek et al.
3-hydroxy diamantane, is produced in high quantity (20.2%). The other metabolites of diamantane represent less than 16% of the total material analysed. The first minor metabolite, 3-diamantanone, is present in the relatively minor amount of about 5%. It is clear, see figure 3B, that two diols, 1,4- and 4,9-di-hydroxy diamantanes, were formed in almost the same amounts (3.0% and 3.4y0, respec- tively). The 3,9-di-hydroxy diamantane (5.0Yo) was produced as a major di- hydroxy derivative, while the 1,6-di-hydroxy diamantane (1.80/,) is only a minor diol. No tri-hydroxy derivatives of diamantane were detected. In the chloroform extract of the reaction mixture, unchanged diamantane (1 5.9% of analysed material) was also found.
In comparison with diamantane, the metabolism of adamantane is more simple. Chromatographic analysis at low temperature (1 10°C) revealed the presence of two mono-hydroxy derivatives, namely, 1 -hydroxy- and 2-hydroxy-adamantane which represent 70.7% and 16.7% respectively. of the material analysed. The ketone, 2- adamantanone, was identified and gave a yield of 5 4 ' j o . Traces of unreacted adamantane were also found ( > 0.5 yo). The high temperature analysis (1 50°C) revealed two major peaks corresponding to the 1,3- and 1,4-di-hydroxy adaman- tane (at relative amount of 11.1 yo and 5.6%, respectively).
Metabolism of diamantane and adamantane by reconstituted cytochrome P-450 system The microsomal system was reconstituted with two cytochrome P-450 isozymes
b and e. Since the catalytic activity of the reconstituted cytochrome P-450 system
c d e h I 4 L L 3 L
0 2 4 6 8 10 12 14 16 18
RETENTION TIME(min)
Figure 4. Gas chromatographv of chloroform extracts of diamantane metabolites from the re-
G.1.c. was at 180 C. T h e identity of peak Z rcniains unresolved as the retention time does not correspond to any available standard; for furthcr symhc,ls see figure 3.
constituted cytochrome P-450b system after 3 h incubation.
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Metabolism of diamantane by P-450 1115
was higher than that of the microsomes, it was necessary to shorten the incubation time to 3 h to get the mono-hydroxy derivatives in the reaction mixture.
Figure 4 shows a typical chromatographic pattern of the diamantane meta- bolites produced by the reconstituted cytochrome P-450b system. There is no detectable unreacted diamantane. Only two mono-hydroxy derivatives, 3- and 4- hydroxy diamantane, were formed in the relative amounts of 1.6% and 51.7%, respectively, of the analysed material. The reconstituted cytochrome P-450 system was also able to generate the diols, 3,9-di-hydroxy-diamantane (10.5%) and 4,9-di- hydroxy-diamantane (24.3%). As in the case of microsomes, the ketone (8% of 3- diamantanone) was found in the reconstituted system.
Almost the same chromatographic profile of the metabolites was obtained when cytochrome P-450e was used instead of the cytochrome P-450b in the reconstituted system.
Adamantane was metabolized by the reconstituted cytochrome P-450b and e system solely to mono-hydroxy derivatives; the amount of the tertiary alcohol (1 - hydroxy adamantane) was approximately seven times higher than that of the secondary one (2-hydroxy adamantane). No unreacted adamantane, ketone or di- hydroxy derivatives were detected.
Discussion The spectral changes caused by diamantane in microsomes from PB-induced
rats, as well as those with cytochrome P-450 isozymes b and e, correspond to Type I described by Schenkman et al. (1981). The spectral studies show that the apparent K6 of diamantane is lower than that of perhydrophenanthrene or adamantane which are known to be the most potent inducers of the Type I spectral change (Anzenba- cher et al. 1984, %pal et al. 1979). Diamantane is considered to be very strongly bound to cytochrome P-450, and is one of the hydrocarbons with the highest affinity for the binding site of cytochrome P-450. Furthermore, the binding of diamantane (or adamantane) to cytochrome P-450 seems to be very specific for cytochrome P-450 isozymes from PB-pretreated animals, as it induces only a minor spectral change when microsomes from BNF-induced rats are used. This implies that diamantane (or adamantane) is able to interact only with some minor isozyme(s) of cytochrome P-450 present in BNF-induced rat liver microsomes. This conclusion is in agreement with the recent knowledge of the architecture of the binding sites of the cytochromes P-450, namely, that non-planar, bulky molecules, e.g. adamantane, are tightly bound by cytochromes P-450 (P-45011) from PB- pretreated animals (White et al. 1984), while cytochromes P-450 induced by BNF (P-450 I) have high affinity for planar structures, as for example polycyclic aromatic hydrocarbons (Lewis et aE. 1986).
Consequently, results provided by spectroscopic experiments are confirmed by metabolic essays. The N-demethylation of aminopyrine by liver microsomes from BNF-pretreated rats is not affected by addition of diamantane or adamantane. In contrast, a significant decrease in the rate of aminopyrine N-demethylation was observed when diamantane or adamantane was added to liver microsomes from PB- induced rats. Comparison of the spectral characteristics leads one to expect that diamantane would be a more potent inhibitor of aminopyrine metabolism than adamantane. This might be caused by the higher hydrophobicity of diamantane and therefore by its stronger binding to the active site of cytochrome P-450.
To obtain a more detailed view of the metabolic fate of the two hydrocarbons
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1116 P. Hodek et al.
used, the metabolism of both compounds by the microsomal MFO system was examined. Studies carried out with liver microsomes from PB- and BNF- pretreated rats provide strong evidence that diamantane (or adamantane) is specifi- cally bound and metabolised by cytochrome P-450 isozymes inducible by pheno- barbital. The analysis of diamantane metabolites produced by the microsomal system from PB-pretreated rats reveals that the apical positions (4 or 9) of the diamantane skeleton are most readily hydroxylated. The relative amount of 4- hydroxy-diamantane is more than twice as high as that of the secondary mono- hydroxy derivatives, e.g. at position 3. According to figure 3A the hydroxylation of medial positions (e.g. 1 or 6) is rare. This may be explained either by steric hindrance of the medial positions or by involvement of a minor cytochrome P-450 isozyme specific for this reaction. As can be expected from the relative amounts of the mono-hydroxy derivatives, the di-hydroxy diamantanes, with the hydroxyl groups in the opposite medial positions (e.g. 1 +6), were found in only small quantities. The relative amounts of the other di-hydroxy diamantanes are similar (see figure 3B). The formation of diamantanone is unexpected; it indicates the possibility of hydroxylation of neighbouring positions, probably secondary and apical ones (e.g. 3 and 4). The identities of the peaks X and Y are unknown, as their retention times do not correspond to any standard available. It is also possible that some tri-hydroxy derivatives may be present in the incubation mixture. This possibility was qualitatively checked by the time-dependence of the disappearance of the parent hydrocarbon (or its hydroxy derivatives) and by the subsequent formation of hydroxylated products in the reaction mixture. Since the tri-hydroxy derivatives are soluble in water, the technique we employed, involving extraction of the metabolic products into chloroform, is not suitable for their detection. Moreover, detection of these compounds is not easy because no appropriate standards were available.
To obtain more information about the role of cytochrome P-450 isozymes b and e in the metabolism of diamantane a reconstituted cytochrome P-450 system of these isozymes was used. Comparison of the chromatographic profiles of extracts from the PB-induced microsomal system, and from reconstituted cytochrome P- 450b system, reveals that in the hydroxylation of diamantane medial positions some isozyme (or even isozymes) distinct from cytochrome P-450 isozyme b, is involved. The same conclusion may be drawn from experiments with cytochrome P-450 isozyme e. Hence, neither cytochrome P-450b nor cytochrome P-450e is responsible for the hydroxylation of the diamantane medial position. The formation of the ketone is a property of both reconstituted cytochrome P-450 systems. In contrast to microsomes, the reconstituted cytochrome P-450 system is much more efficient in the metabolism of diamantane. After 3 h there is no detectable amount of unreacted diamantane remaining in the reaction mixture, and after a further period of 3 h incubation, the mono-hydroxy derivatives are also fully metabolized. In the case of microsomes, the unreacted diamantane and mono-hydroxy derivatives can be detected even after 6 h.
Our findings concerning the metabolism of adamantane by the reconstituted cytochrome P-450b system are in agreement with the results obtained by White et al. (1984) who also studied the metabolic fate of that compound in a reconstituted cytochrome P-450 system, using isozyme LM, (purified from liver microsomes of phenobarbital-pretreated rabbits). Neither keto- nor di-hydroxy adamantanes were found in either of the reconstituted systems, in contrast to the findings with
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Metabolism of diamantane by P-450 1117
microsomes were both ketone and di-hydroxy derivatives were detected. No explanation of these differences has been given, to the present.
In summary, data presented show that diamantane is a novel specific and highly efficient substrate of the cytochromes P-450 inducible by phenobarbital. In liver microsomes of phenobarbital-pretreated rats the cytochromes P-450 bind diaman- tane very tightly and form a complex which is characterized by one of the lowest values of the K: known.
From comparison of the patterns of metabolism of diamantane in liver micro- somes from PB-pretreated rats, and in the reconstituted cytochrome P-450b and e systems, follows that the hydroxylation of the medial position of diamantane is caused by some other isozyme than those mentioned above. The formation of apical and secondary hydroxy derivatives is catalysed efficiently by both phenobarbital- inducible isozymes of cytochrome P-450. The cytochrome P-450 isozymes indu- cible by 8-naphthoflavone are not capable either of binding or of metabolising diamantane (and adamantane).
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1118 Metabolism of diamantane by P-450
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