Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus H. Nau, K. Brendel & C. Liddiard To cite this article: H. Nau, K. Brendel & C. Liddiard (1979) Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus, Drug Metabolism Reviews, 9:1, 131-146, DOI: 10.3109/03602537909046436 To link to this article: http://dx.doi.org/10.3109/03602537909046436
Published online: 22 Sep 2008.
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DRUG METABOLISM REVIEWS, 9(1), 131-146 (1979)
Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus* H. NAU, K. BRENDEL,t and C. LIDDIARD Institut fiir Toxikologie und Embryonalpharmakologie Freie Universitat D-1000 Berlin 33, West Germany
I. 11.
111.
INTRODUCTION
.....................................
......................
EXPERIMENTAL PROCEDURES A. Biological Material B. Preparation of Fetal Liver Cell Cultures C. GC and GC-MS Analyses
.............................. ........... .......................... EXPERIMENTAL RESULTS AND DISCUSSION ..........
................................... .........................................
132 133 133 133 134 136
Acknowledgments
143
References
145
*Presented at Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the auspices of the American Society for Pharmacology and Experimental Therapeutics. t P r e s e n t address: Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona 85724.
131 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
132
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I. INTRODUCTION
The average number of drugs taken by women during pregnancy was found to be between 3 and 10 in several studies; moreover, up to 80% of the drugs ingested were not prescribed by a physician [1-3]. In addition, exposure t o nicotine, cosmetic products, artificial sweeteners, and environmental contaminants such a s pesticides and many other substances must be considered, Since it i s well established that the majority of compounds readily cross the placenta [4], the human embryo and fetus is exposed throughout gestation to a large number of xenobiotic substances. Little is known about the possible harmful effects of these compounds on the development of the human embryo and fetus, and wide variety of techniques in prenatal toxicology i s needed for the evaluation of embryotoxicity and teratogenicity [51. Once these compounds pass the placenta and enter the fetal circulation, they may be metabolized by enzyme systems which a r e known to be present in the fetus, particularly in liver and adrenal gland [681. Since metabolites a r e often more polar than the parent compounds, placental diffusion back into the maternal circulation i s slower and thus accumulation in the fetus may occur. Potentially toxic metabolites may also be produced which, owing to their short half-lives, may otherwise not reach the fetus. Until now, the fetal metabolism of only a few drugs has been studied in human fetal liver microsomes. These include chlorpromazine, hexobarbital, meperidine, and aminopyrine by Pelkonen [ 9, 101. desmethylimipramine by Bahr e t al. [ I l l , ethylmorphine by Rane and Ackermann [12], and diazepam by Idanpaan-Heikkila et al. [13] and Ackermann and Richter [14]; for a review, s e e Waddell and Marlowe [El. In all of these investigations, an isolated microsomal enzyme system was used. A f t e r preliminary work which we had done with isolated suspended hepatocytes of rat and rabbit, we decided to test the feasibility of using primary liver cell culture for the study of human fetal drug metabolism. Although Pelkonen et al. [ 16, 173 have investigated the metabolism of benzo(a)pyrene in primary monolayer culture from human fetal liver cells, the metabolism of drugs in such systems has not yet been reported. In this communication we describe our initial results with suspension cultures of isolated human fetal liver cells as well a s with cultures of liver fragments in regard to their ability to metabolize the benzodiazepines: prazepam, diazepam, and medazepam. In order
HUMAN FETAL HEPATOCYTE CULTURES
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to detect very low levels of metabolism we employed gas chromatography with electron capture detection a s a highly sensitive and selective analytical technique. The sensitivity and specificity of the analysis were further enhanced by using a directly coupled gas chromatograph-mass spectrometer-computer system (GC-MS-computer) with selected ion monitoring capability (mass fragmentography) [ 1 8 ] .
11.
EXPERIMENTAL PROCEDURES A.
Biological Material
Fetuses were obtained by either hysterotomy, prostaglandin-induction, or curettage, Hysterotomy is the method of choice since the fetus is obtained in toto and the experimental conditions can be rigorously controlled. However, hysterotomy i s rarely performed in our countries and fetuses could not be obtained in sufficient numbers by this technique. Prostaglandin-induction did not result in reliable fetal material. One reason may be that the process of expulsion of the fetus is of several hours duration and cannot be controlled. Furthermore, fetal death may occur during the induction period several hours before the expulsion of the fetus [19], thus rendering fetal tissues unsuitable for biochemical studies. Although curettage usually yields fetal fragments only, it was often possible to identify the fetal liver within the trunk and sample it under sterile conditions. Regardless of the procedure used, the livers were excised from the fetuses a s soon a s possible and kept in Dulbecco's buffer a t O°C until preparation of the cells began (usually within 0.5 to 3 hr). B. Preparation of Fetal Liver Cell Cultures All preparations were done under stringent sterile conditions in a laminar flow hood. Small liver pieces were obtained either by pressing fetal liver through a nylon sieve in Dulbecco's buffer (Cazf-free) o r by mincing o r slicing with scalpels; all methods yielded comparable results. The pieces were washed several times and then incubated with Dispase I [20] (1 mg enzyme in 1 0 ml buffer; 0.6 ml of this solution was used per 100 mg liver for 30 to 120 min at 37'C with shaking). After incubation, the mixture was filtered through a 250-km mesh sieve to remove undigested material and the resulting filtrate was centrifuged
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at 200 rpm and 4OC for 10 min. The supernatant fluid was decanted and the cellular pellet was resuspended i n Ham's F-12 medium and preincubated at 37OC for 30 min. Then the mixture was filtered through a 100-pm mesh sieve and centrifuged at 200 rpm at 4OC. The resulting cellular pellet was resuspended in buffer and centrifuged again. The final cellular pellet was suspended in Ham's F-12 medium fortified with 15% fetal calf serum and glutamine (2 mM final concentration) and protected against bacterial growth by addition of penicillin (10 E/ml) and streptomycin (10 p g/ml). Then the drug under investigation was added with a final concentration of 1 to 20 pg/ml. Yield and viability of the cells were determined in a Neubauer counting chamber using the trypan blue dye exclusion technique. The morphological appearance of the cells during the incubation period was monitored by phase-contrast light microscopy and electron microscopy (details to be published). Incubation flasks for cell culturing were siliconized 15 ml groundjoint Erlenmeyer flasks with sedimentary bottoms, The ground-joint glass stoppers were modified to admit a strand of silicone rubber tubing with inlet and outlet. This constituted a closed system for exchange of 0, and CO, from which water vapor could not escape, thus avoiding concentration changes during prolonged incubation (Fig. I), Incubation was performed on a gyratory shaker (60 gyrations per min) which was housed in a 37'C incubator up to 65 hr following addition of the drug, Samples were removed at selected time intervals, sonicated, and frozen until extraction. Organ cultures of small pieces of fetal liver were prepared by pressing the liver through a nylon sieve and washing the resulting liver fragments several times. Incubation was performed in the same manner a s described above for Dispase-isolated liver cells. C. GC and GC-MS Analvses
To 50 to 200 p l of liver cells or organ suspension (which initially contained 0.1 to 2 p g of the drug), the internal standard (see below) was added; the samples were extracted with 1 ml ethyl acetate (Nanograde, Mallinckrodt) on a vortex mixer (2 min). After brief centrifugation (2 min), 800 p l of the organic phase was removed and evaporated with nitrogen at room temperature. The dried residues were redissolved in 30 p l pyridine, and 1 pl was injected into a Carlo Erba 2300 gas chromatograph equipped with an electron capture detector (60 ng diazepam was used a s internal standard if prazepam metabolites were to be measured and
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ground joint with silicone tube
8
rubber cap (sampling)
Incubation flask FIG, 1. Incubation flask with silicone rubber oxygenator in ground joint stopper for long-time incubations.
vice versa). For the accurate quantitation of hydroxylated metabolites, the samples were trimethylsilylated a t 5OoC for 30 min by the addition of 30 pl bis-trimethylsilyl trifluoracetamide/l% trimethylchlorosilane (Regis Co.), and 2 p l of the resulting mixture was analyzed. The GC-MS-computer system consisted of a Perkin-Elmer F-22 gas chromatograph directly coupled via a one-stage Watson-Biemann separator to the ion source of a CH 7 A Varian MAT mass spectrometer. A Varian SS-100 data system was used for acquisition of the GC-MS data (for identification of metabolites) and selected ion monitoring by switching the accelerating voltage (for quantitation of the metabolites). Methyl-d, diazepam was used a s i.s. (60 ng) for the quantitation of all benzodiazepine metabolites studied.
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The methods described have sufficient sensitivity to detect and quantitate very low levels of benzodiazepine metabolites (go% exclude trypan blue). The fetal liver cells were routinely checked during the incubation period by phasecontrast light microscopy. When prazepam (Demetrin) was added to fetal liver organ culture (human fetus 10 weeks of gestation), and an extract of the incubation medium was analyzed by gas chromatography, the chromatogram shown in Fig, 2A resulted. In addition to the peak corresponding to the parent drug, other peaks which indicated the formation of metabolites were present. Since an electron capture detector was used, which selectively responds to halogen-containing compounds, benzodiazepines and their metabolites (all investigated drugs contained one chlorine atom) are detected with high sensitivity and selectivity while suppressing all endogenous compounds of the liver as well a s constituents of the medium. Identical cultures, but without liver cells o r with nonviable liver cells, did not produce any metabolites (Fig. 2B). Identification of the metabolites was accomplished with a GCMS-computer system and mass spectra were obtained which indicated that prazepam (I) was transformed by dealkylation into (II) and by 3-hydroxylation into (111); oxazepam (IV) i s formed either by 3-hydroxylation of (11) o r dealkylation of 411); see Scheme 1. The metabolites-after trimethylsilylation to enhance the volatility of hydroxylated compounds present-were quantitated by GC-MS using selected ion monitoring (Fig. 3) of m a s s 342 (metabolite 11), 324 (Prazepam, I), 383 (metabolite 111), and 429 (metabolite IV). Incubation of prazepam with an organ culture containing small pieces of fetal liver (Fig. 4A)yielded a similar pattern of metabolites as incubation with cultures of isolated human fetal liver cells (Fig. 4B).
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meanethylDiazepam
A
3sfydroxyFmzepam
FIG. 2. Electron capture gas chromatogram (isotherm, 26OoC) of an ethyl acetate extract of incubations containing prazepam. A: With isolated fetal liver cells (fetal age: 10 weeks), B: Without liver cells, The relationship between metabolite appearance and incubation time with prazepam is shown in Fig. 5, indicating that even after 60 h r significant activity is retained in the liver cell culture. After 24 h r , 0.88 nmol of prazepam was metabolized per l o 6 cells. Averaged, this equals transformation of 36 pmol prazepam/106 cells/hr.
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HUMAN FETAL HEPATOCYTE CULTURES
Prazepam
3-Hydroxy prazepam
139
N-Deemethyl diazepam
Oxazepam
SCHEME 1. Metabolism of prazepam.
These results indicate that prazepam was extensively metabolized in the liver culture of the fetus of early gestation by 3-hydroxylation into 3-hydroxy prazepam (III) and desalkylation into desmethyl diazepam 01). Both metabolites appeared in approximately equal amounts, while their further transformation into oxazepam (IV) was much less pronounced (Fig. 5). All of the fetal metabolites identified were previously found by Di Carlo et al. [24] in adult humans in vivo (I1 in blood, 111 and IV a s glucuronides in urine). However, (III) was not found a s metabolite in adult human liver microsomes [25]. We a r e at present investigating the cause of this apparent discrepancy between the prazepam metabolism of the adult and fetal human in vitro systems. Selected ion monitoring a s well as electron capture gas chromatography were also used for the detection of diazepam (Valium)
NAU, BRENDEL, AND LIDDIARD
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140
3-Hydr oxy prazepam
m/e 383 1 " I " l
x2
I
0
-
L
I "
1
1
2
l " l "
,"I
mle342
3
4 MINUTES
' '
" I
" 1
5
6
1 1 -
1
8
814/ MI/ 104 FIG. 3. Selected ion monitoring (isotherm, 260' C) of an ethyl acetate extract of incubations containing prazepam and isolated human fetal liver cells (fetal age: 10 weeks).
metabolites in human fetal liver cells and organ cultures. This drug was also metabolized, though to a lesser extent, both by dealkylation to form desmethyl diazepam and by 3-hydroxylation to yield 3-hydroxy diazepam (Fig. 6). It was recently shown by Ackermann and Richter [14] that both metabolites were also formed by human fetal liver microsomes. Medazepam (Nobrium) was incubated in the liver culture of a 20week old fetus, and N-desmethyl medazepam a s well a s diazepam (oxidation in the 2-position of the diazepin ring) were identified a s metabolites (Fig. 7A). Following trimethylsilylation, 2-hydroxy medazepam and 3-hydroxy diazepam were also found (Fig. 7B). Further studies will show the full extent to which these cultures of human fetal liver cells can be exploited to simulate hepatic drug
v
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I
A
m/@287
+ J l
-
1 " l " I " l " I " ~ " 1 2 3 4
5
6
7
8
9
10
11
B
1
2
3
4
5
6
7
8
9
1 0 1 1
MINUTES
FIG. 4. Selected ion monitoring (linear temperature programming 200 to 26OoC with 10°C/min) of prazepam metabolites formed in (A) human fetal liver organ culture and (B) isolated human fetal liver cells. Methyl-d3 diazepam was internal standard (ion 287).
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Incubation time
"
FIG. 5. Plot of nmole prazepam metabolites formed per lo6 human fetal liver cells vs time; 45 X l o 6 cells and 62 nmole prazepam/ ml incubation mixture. (A) 3-Hydroxy prazepam (III). (0)Desmethyl diazepam (11). (0)Oxazepam (IV).
metabolisms of the human embryo and fetus. Advantages a s compared to the microsomal enzyme systems include that cell cultures do not require cofactors and remain viable for several days. Thus minor metabolites may also be produced in sufficient quantity for their detection. Furthermore, intact cells are functional in our systems rather than isolated cell organelles. This may allow us to investigate pathways of reactions between drugs and/or their metabolites o r short-lived intermediates with cellular constituents outside the endoplasmic reticulum.
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HUMAN FETAL HEPATOCYTE CULTURES
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t;
qlSH3
Ct
0
OT Ms
A
FIG. 6. Selected ion monitoring of trimethylsilylated diazepam metabolites (desmethyl diazepam and 3 - h y d r o 3 diazepam) formed in a culture of human fetal liver fragments (fetus: 24 weeks of gestation).
Acknowledgments The donation of Dispase by Drs. J. Flach and J. Bauer (Boehringer, Mannheim), of methyl d3-diazepam by Dr. W. Vetter, (Hoffmann LaRoche, Basel), and of 3-hydroxy prazepam by Dr. K.-0. Vollmer (Godecke, Freiburg), the excellent assistance of Mr. W. Wittfoht, Ms. E. Schulz, D. Jesdinsky, and the help of Ms. N. Nau in the preparation of this manuscript a r e gratefully acknowledged. We should like t o express our gratitude to Profs. D. Neubert and H. -J. Merker and Dr. T. Kwasigroch for their helpful advice and discussions.
144
NAU, BRENDEL, AND LIDDIARD H
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I
A
C
L0 3
t c
D2 W
I-
z
H
B
1
2
3
4
5 6 MINUTES
7
8
9
FIG. 7. Selected ion monitoring of medazepam metabolites formed in a culture of human fetal liver fragments (fetus: 24 weeks of gestation). A : Direct injection of ethyl acetate extraction indicating desmethyl medazepam and diazepam. B: Injection after silylation of Sample A indicating 2-hydroxy medazepam and 3-hydroxy diazepam.
HUMAN FETAL HEPATOCYTE CULTURES
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We a l s o thank Dr. J. Lange, Klinikum Charlottenburg, and Dr. V. Haase, Klinikum Steglitz, without whose cooperation this work would not have been possible. This work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 29.
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REFERENCES
[11 R. M. Hill, Clin. Pharmacol. Ther., 1_4, 654-659 (1973). [ 2 ] L. Finnegan, Ann. N. Y. Acad. Sci., 281, 74-89 (1976). [3] G. Boethius, Eur. J. Clin. Pharmacol., 12, 37-43 (1977). [4] B. L. Mirkin and S. Singh, in Perinatal Pharmacology and Therapeutics (B. L. Mirkin, ed.), Academic, New York, 1976, pp. 1-70. [5] D. Neubert, H.-J. Merker, and T. E. Kwasigroch (eds.), Methods in Prenatal Toxicology, Thieme, Stuttgart, 1977. [6] S. J. Yaffe, A. Rane, F. Sjoqvist, L. 0. Boreus, and S. Orrenius, L i f e i . , _9, 1189-1200 (1970). [ 7 ] 0. Pelkonen and J. T. Karki, Acta Pharmacol. Toxicol., 30, 158-160 (1971). [8] M. R. Juchau and M. G . Pedersen, Life Sci., 12, 193-204 (1973). [9] 0.Pelkonen, M. Vorne, P. Jouypila, and N. T. Karki, 4 Pharmacol. Toxicol., 2,284-294 (1971). [lo] 0. Pelkonen, E. H. Kaltiala, T. K. I. Larmi, and N . T. Karki, Clin. Pharmacol. Ther., 12, 840-846 (1973). [ll] C. von Bahr, A. Rane, S. Orrenius, and F. Sjoqvist, &&a Pharmacol. Toxicol., 2, 58-64 (1974). [12] A. Rane and E. Ackermann, Clin. Pharmacol. Ther., 2, 663670 (1972). [13] J. F. Idanpaan-Heikkila, P. I. Jouppila, J. 0. Puolakka, and MS :. Vorne, Am. J. Obstet. Gynecol., 109, 1011-1016 (1971). [14] E. Ackermann and K. Richter, Eur. J. Clin. Pharmacol., 11, 43-49 (1977). [ 151 W. J. Waddell and G. C. Marlowe, in Perinatal Pharmacology and Therapeutics (E. L. Mirkin, ed.), Academic, New York, 1976, pp. 119-269. [16] 0. Pelkonen, P. Korhonen, P. Jouppila, and N. Karki, Life Sci., -16, 1403-1410 (1975). -
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0. Pelkonen, i n Active Intermediates: Formation Toxicity and Inactivation (R. L. Snyder, D. J. Jollow, J. J. Kocsis, and H. Vainio, e d s . ) , Plenum, New York, 1977, pp. 148-159. H. Nau, C. Liddiard, K. Brendel, W. Wittfoht, and J. Lange, in M a s s Spectrometry and Combined Techniques in Medicine, Clinical C h e m i s t r y and Clinical Biochemistry (M. Eggstein and H. M. Liebich, e d s . ) , (Proceedings of the Symposium in Tubingen, G. F. R., November 1977), pp. 346-358. I. S. F r a s e r , K. M. Nicholson, G. G r a h a m , and H. Boyle, Prostaglandins, 1_3(6), 1161- 1177 (1977). T. Takaoka, S. Yasumoto, and H. Katsota, Japan. J. Exp. 45, 317-326 (1975). M. Kekomaki, M. Seppala, C. Ehnholm, A. L. Schwartz, and K. Raivio, Int. J. C a n c e r , !, 250-258 (1971). M. N. B e r r y and D. S. Friend, J. Cell. Biol., 43, 506-520 (1969). P. 0. Seglen, i n Methods in C e l l Biology, Vol. 13 (D. M. P r e s c o t t , ed.), Academic, New York, 1976, pp. 29-83. F. J. Di Carlo, J.-P. Viau, J. E. Epps, and L. J. Haynes, Clin. P h a r m a c o l . T h e r . , 890-897 (1970). J.-P. Viau, J. E. Epps, and F. J. Di Carlo, Biochem. Pharmacol., 21, 563-569 (1972).
e.,
11,
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus H. Nau, K. Brendel & C. Liddiard To cite this article: H. Nau, K. Brendel & C. Liddiard (1979) Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus, Drug Metabolism Reviews, 9:1, 131-146, DOI: 10.3109/03602537909046436 To link to this article: http://dx.doi.org/10.3109/03602537909046436
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 5
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [Deakin University Library]
Date: 05 November 2015, At: 18:06
Downloaded by [Deakin University Library] at 18:06 05 November 2015
DRUG METABOLISM REVIEWS, 9(1), 131-146 (1979)
Benzodiazepine Metabolism in Cultures of Isolated Hepatocytes and Liver Fragments of Human Fetus* H. NAU, K. BRENDEL,t and C. LIDDIARD Institut fiir Toxikologie und Embryonalpharmakologie Freie Universitat D-1000 Berlin 33, West Germany
I. 11.
111.
INTRODUCTION
.....................................
......................
EXPERIMENTAL PROCEDURES A. Biological Material B. Preparation of Fetal Liver Cell Cultures C. GC and GC-MS Analyses
.............................. ........... .......................... EXPERIMENTAL RESULTS AND DISCUSSION ..........
................................... .........................................
132 133 133 133 134 136
Acknowledgments
143
References
145
*Presented at Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the auspices of the American Society for Pharmacology and Experimental Therapeutics. t P r e s e n t address: Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona 85724.
131 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
132
NAU, BRENDEL, AND LIDDIARD
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I. INTRODUCTION
The average number of drugs taken by women during pregnancy was found to be between 3 and 10 in several studies; moreover, up to 80% of the drugs ingested were not prescribed by a physician [1-3]. In addition, exposure t o nicotine, cosmetic products, artificial sweeteners, and environmental contaminants such a s pesticides and many other substances must be considered, Since it i s well established that the majority of compounds readily cross the placenta [4], the human embryo and fetus is exposed throughout gestation to a large number of xenobiotic substances. Little is known about the possible harmful effects of these compounds on the development of the human embryo and fetus, and wide variety of techniques in prenatal toxicology i s needed for the evaluation of embryotoxicity and teratogenicity [51. Once these compounds pass the placenta and enter the fetal circulation, they may be metabolized by enzyme systems which a r e known to be present in the fetus, particularly in liver and adrenal gland [681. Since metabolites a r e often more polar than the parent compounds, placental diffusion back into the maternal circulation i s slower and thus accumulation in the fetus may occur. Potentially toxic metabolites may also be produced which, owing to their short half-lives, may otherwise not reach the fetus. Until now, the fetal metabolism of only a few drugs has been studied in human fetal liver microsomes. These include chlorpromazine, hexobarbital, meperidine, and aminopyrine by Pelkonen [ 9, 101. desmethylimipramine by Bahr e t al. [ I l l , ethylmorphine by Rane and Ackermann [12], and diazepam by Idanpaan-Heikkila et al. [13] and Ackermann and Richter [14]; for a review, s e e Waddell and Marlowe [El. In all of these investigations, an isolated microsomal enzyme system was used. A f t e r preliminary work which we had done with isolated suspended hepatocytes of rat and rabbit, we decided to test the feasibility of using primary liver cell culture for the study of human fetal drug metabolism. Although Pelkonen et al. [ 16, 173 have investigated the metabolism of benzo(a)pyrene in primary monolayer culture from human fetal liver cells, the metabolism of drugs in such systems has not yet been reported. In this communication we describe our initial results with suspension cultures of isolated human fetal liver cells as well a s with cultures of liver fragments in regard to their ability to metabolize the benzodiazepines: prazepam, diazepam, and medazepam. In order
HUMAN FETAL HEPATOCYTE CULTURES
133
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to detect very low levels of metabolism we employed gas chromatography with electron capture detection a s a highly sensitive and selective analytical technique. The sensitivity and specificity of the analysis were further enhanced by using a directly coupled gas chromatograph-mass spectrometer-computer system (GC-MS-computer) with selected ion monitoring capability (mass fragmentography) [ 1 8 ] .
11.
EXPERIMENTAL PROCEDURES A.
Biological Material
Fetuses were obtained by either hysterotomy, prostaglandin-induction, or curettage, Hysterotomy is the method of choice since the fetus is obtained in toto and the experimental conditions can be rigorously controlled. However, hysterotomy i s rarely performed in our countries and fetuses could not be obtained in sufficient numbers by this technique. Prostaglandin-induction did not result in reliable fetal material. One reason may be that the process of expulsion of the fetus is of several hours duration and cannot be controlled. Furthermore, fetal death may occur during the induction period several hours before the expulsion of the fetus [19], thus rendering fetal tissues unsuitable for biochemical studies. Although curettage usually yields fetal fragments only, it was often possible to identify the fetal liver within the trunk and sample it under sterile conditions. Regardless of the procedure used, the livers were excised from the fetuses a s soon a s possible and kept in Dulbecco's buffer a t O°C until preparation of the cells began (usually within 0.5 to 3 hr). B. Preparation of Fetal Liver Cell Cultures All preparations were done under stringent sterile conditions in a laminar flow hood. Small liver pieces were obtained either by pressing fetal liver through a nylon sieve in Dulbecco's buffer (Cazf-free) o r by mincing o r slicing with scalpels; all methods yielded comparable results. The pieces were washed several times and then incubated with Dispase I [20] (1 mg enzyme in 1 0 ml buffer; 0.6 ml of this solution was used per 100 mg liver for 30 to 120 min at 37'C with shaking). After incubation, the mixture was filtered through a 250-km mesh sieve to remove undigested material and the resulting filtrate was centrifuged
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at 200 rpm and 4OC for 10 min. The supernatant fluid was decanted and the cellular pellet was resuspended i n Ham's F-12 medium and preincubated at 37OC for 30 min. Then the mixture was filtered through a 100-pm mesh sieve and centrifuged at 200 rpm at 4OC. The resulting cellular pellet was resuspended in buffer and centrifuged again. The final cellular pellet was suspended in Ham's F-12 medium fortified with 15% fetal calf serum and glutamine (2 mM final concentration) and protected against bacterial growth by addition of penicillin (10 E/ml) and streptomycin (10 p g/ml). Then the drug under investigation was added with a final concentration of 1 to 20 pg/ml. Yield and viability of the cells were determined in a Neubauer counting chamber using the trypan blue dye exclusion technique. The morphological appearance of the cells during the incubation period was monitored by phase-contrast light microscopy and electron microscopy (details to be published). Incubation flasks for cell culturing were siliconized 15 ml groundjoint Erlenmeyer flasks with sedimentary bottoms, The ground-joint glass stoppers were modified to admit a strand of silicone rubber tubing with inlet and outlet. This constituted a closed system for exchange of 0, and CO, from which water vapor could not escape, thus avoiding concentration changes during prolonged incubation (Fig. I), Incubation was performed on a gyratory shaker (60 gyrations per min) which was housed in a 37'C incubator up to 65 hr following addition of the drug, Samples were removed at selected time intervals, sonicated, and frozen until extraction. Organ cultures of small pieces of fetal liver were prepared by pressing the liver through a nylon sieve and washing the resulting liver fragments several times. Incubation was performed in the same manner a s described above for Dispase-isolated liver cells. C. GC and GC-MS Analvses
To 50 to 200 p l of liver cells or organ suspension (which initially contained 0.1 to 2 p g of the drug), the internal standard (see below) was added; the samples were extracted with 1 ml ethyl acetate (Nanograde, Mallinckrodt) on a vortex mixer (2 min). After brief centrifugation (2 min), 800 p l of the organic phase was removed and evaporated with nitrogen at room temperature. The dried residues were redissolved in 30 p l pyridine, and 1 pl was injected into a Carlo Erba 2300 gas chromatograph equipped with an electron capture detector (60 ng diazepam was used a s internal standard if prazepam metabolites were to be measured and
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ground joint with silicone tube
8
rubber cap (sampling)
Incubation flask FIG, 1. Incubation flask with silicone rubber oxygenator in ground joint stopper for long-time incubations.
vice versa). For the accurate quantitation of hydroxylated metabolites, the samples were trimethylsilylated a t 5OoC for 30 min by the addition of 30 pl bis-trimethylsilyl trifluoracetamide/l% trimethylchlorosilane (Regis Co.), and 2 p l of the resulting mixture was analyzed. The GC-MS-computer system consisted of a Perkin-Elmer F-22 gas chromatograph directly coupled via a one-stage Watson-Biemann separator to the ion source of a CH 7 A Varian MAT mass spectrometer. A Varian SS-100 data system was used for acquisition of the GC-MS data (for identification of metabolites) and selected ion monitoring by switching the accelerating voltage (for quantitation of the metabolites). Methyl-d, diazepam was used a s i.s. (60 ng) for the quantitation of all benzodiazepine metabolites studied.
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The methods described have sufficient sensitivity to detect and quantitate very low levels of benzodiazepine metabolites (go% exclude trypan blue). The fetal liver cells were routinely checked during the incubation period by phasecontrast light microscopy. When prazepam (Demetrin) was added to fetal liver organ culture (human fetus 10 weeks of gestation), and an extract of the incubation medium was analyzed by gas chromatography, the chromatogram shown in Fig, 2A resulted. In addition to the peak corresponding to the parent drug, other peaks which indicated the formation of metabolites were present. Since an electron capture detector was used, which selectively responds to halogen-containing compounds, benzodiazepines and their metabolites (all investigated drugs contained one chlorine atom) are detected with high sensitivity and selectivity while suppressing all endogenous compounds of the liver as well a s constituents of the medium. Identical cultures, but without liver cells o r with nonviable liver cells, did not produce any metabolites (Fig. 2B). Identification of the metabolites was accomplished with a GCMS-computer system and mass spectra were obtained which indicated that prazepam (I) was transformed by dealkylation into (II) and by 3-hydroxylation into (111); oxazepam (IV) i s formed either by 3-hydroxylation of (11) o r dealkylation of 411); see Scheme 1. The metabolites-after trimethylsilylation to enhance the volatility of hydroxylated compounds present-were quantitated by GC-MS using selected ion monitoring (Fig. 3) of m a s s 342 (metabolite 11), 324 (Prazepam, I), 383 (metabolite 111), and 429 (metabolite IV). Incubation of prazepam with an organ culture containing small pieces of fetal liver (Fig. 4A)yielded a similar pattern of metabolites as incubation with cultures of isolated human fetal liver cells (Fig. 4B).
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meanethylDiazepam
A
3sfydroxyFmzepam
FIG. 2. Electron capture gas chromatogram (isotherm, 26OoC) of an ethyl acetate extract of incubations containing prazepam. A: With isolated fetal liver cells (fetal age: 10 weeks), B: Without liver cells, The relationship between metabolite appearance and incubation time with prazepam is shown in Fig. 5, indicating that even after 60 h r significant activity is retained in the liver cell culture. After 24 h r , 0.88 nmol of prazepam was metabolized per l o 6 cells. Averaged, this equals transformation of 36 pmol prazepam/106 cells/hr.
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Prazepam
3-Hydroxy prazepam
139
N-Deemethyl diazepam
Oxazepam
SCHEME 1. Metabolism of prazepam.
These results indicate that prazepam was extensively metabolized in the liver culture of the fetus of early gestation by 3-hydroxylation into 3-hydroxy prazepam (III) and desalkylation into desmethyl diazepam 01). Both metabolites appeared in approximately equal amounts, while their further transformation into oxazepam (IV) was much less pronounced (Fig. 5). All of the fetal metabolites identified were previously found by Di Carlo et al. [24] in adult humans in vivo (I1 in blood, 111 and IV a s glucuronides in urine). However, (III) was not found a s metabolite in adult human liver microsomes [25]. We a r e at present investigating the cause of this apparent discrepancy between the prazepam metabolism of the adult and fetal human in vitro systems. Selected ion monitoring a s well as electron capture gas chromatography were also used for the detection of diazepam (Valium)
NAU, BRENDEL, AND LIDDIARD
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140
3-Hydr oxy prazepam
m/e 383 1 " I " l
x2
I
0
-
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,"I
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metabolites in human fetal liver cells and organ cultures. This drug was also metabolized, though to a lesser extent, both by dealkylation to form desmethyl diazepam and by 3-hydroxylation to yield 3-hydroxy diazepam (Fig. 6). It was recently shown by Ackermann and Richter [14] that both metabolites were also formed by human fetal liver microsomes. Medazepam (Nobrium) was incubated in the liver culture of a 20week old fetus, and N-desmethyl medazepam a s well a s diazepam (oxidation in the 2-position of the diazepin ring) were identified a s metabolites (Fig. 7A). Following trimethylsilylation, 2-hydroxy medazepam and 3-hydroxy diazepam were also found (Fig. 7B). Further studies will show the full extent to which these cultures of human fetal liver cells can be exploited to simulate hepatic drug
v
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I
A
m/@287
+ J l
-
1 " l " I " l " I " ~ " 1 2 3 4
5
6
7
8
9
10
11
B
1
2
3
4
5
6
7
8
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FIG. 4. Selected ion monitoring (linear temperature programming 200 to 26OoC with 10°C/min) of prazepam metabolites formed in (A) human fetal liver organ culture and (B) isolated human fetal liver cells. Methyl-d3 diazepam was internal standard (ion 287).
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Incubation time
"
FIG. 5. Plot of nmole prazepam metabolites formed per lo6 human fetal liver cells vs time; 45 X l o 6 cells and 62 nmole prazepam/ ml incubation mixture. (A) 3-Hydroxy prazepam (III). (0)Desmethyl diazepam (11). (0)Oxazepam (IV).
metabolisms of the human embryo and fetus. Advantages a s compared to the microsomal enzyme systems include that cell cultures do not require cofactors and remain viable for several days. Thus minor metabolites may also be produced in sufficient quantity for their detection. Furthermore, intact cells are functional in our systems rather than isolated cell organelles. This may allow us to investigate pathways of reactions between drugs and/or their metabolites o r short-lived intermediates with cellular constituents outside the endoplasmic reticulum.
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t;
qlSH3
Ct
0
OT Ms
A
FIG. 6. Selected ion monitoring of trimethylsilylated diazepam metabolites (desmethyl diazepam and 3 - h y d r o 3 diazepam) formed in a culture of human fetal liver fragments (fetus: 24 weeks of gestation).
Acknowledgments The donation of Dispase by Drs. J. Flach and J. Bauer (Boehringer, Mannheim), of methyl d3-diazepam by Dr. W. Vetter, (Hoffmann LaRoche, Basel), and of 3-hydroxy prazepam by Dr. K.-0. Vollmer (Godecke, Freiburg), the excellent assistance of Mr. W. Wittfoht, Ms. E. Schulz, D. Jesdinsky, and the help of Ms. N. Nau in the preparation of this manuscript a r e gratefully acknowledged. We should like t o express our gratitude to Profs. D. Neubert and H. -J. Merker and Dr. T. Kwasigroch for their helpful advice and discussions.
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I
A
C
L0 3
t c
D2 W
I-
z
H
B
1
2
3
4
5 6 MINUTES
7
8
9
FIG. 7. Selected ion monitoring of medazepam metabolites formed in a culture of human fetal liver fragments (fetus: 24 weeks of gestation). A : Direct injection of ethyl acetate extraction indicating desmethyl medazepam and diazepam. B: Injection after silylation of Sample A indicating 2-hydroxy medazepam and 3-hydroxy diazepam.
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We a l s o thank Dr. J. Lange, Klinikum Charlottenburg, and Dr. V. Haase, Klinikum Steglitz, without whose cooperation this work would not have been possible. This work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 29.
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REFERENCES
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e.,
11,
