Abstract
Evidence has been presented for auto-induced human cytochrome P450 3A enzyme involvement in the teratogenicity and clinical outcome of thalidomide due to oxidation to 5-hydroxythalidomide and subsequent metabolic activation in livers. In this study, more relevant human placenta preparations and placental BeWo cells showed low but detectable P450 3A4/5 mRNA expression and drug oxidation activities. Human placental microsomal fractions from three subjects showed detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. Human placental BeWo cells, cultured in the recommended media, also indicated detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. To reduce any masking effects by endogenous hormones used in the recommended media, induction of P450 3A4/5 mRNA and oxidation activities were measured in placental BeWo cells cultured with a modified medium containing 5% charcoal-stripped fetal bovine serum. Thalidomide significantly induced P450 3A4/3A5, 2B6, and pregnane X receptor (PXR) mRNA levels 2 to 3-fold, but rifampicin only enhanced P450 3A5 and PXR mRNA under the modified media conditions. Under these modified conditions, thalidomide also significantly induced midazolam 1´-hydroxylation and thalidomide 5-hydroxylaion activities 3-fold but not bupropion hydroxylation activity. Taken together, activation of thalidomide to 5-hydroxythalidomide with autoinduction of P450 3A enzymes in human placentas, as well as livers, is suggested in vivo.
Keywords: placenta, rifampicin, phenobarbital, drug oxidation, induction
Introduction
Thalidomide has been proposed to exert its pharmacological and toxic actions in primates through multiple mechanisms. The oxidative metabolism of thalidomide is important for both its teratogenicity and anticancer efficacy (Gordon et al., 1981). Thalidomide induced limb abnormalities in whole-embryos of transchromosomic mice containing a human cytochrome P450 3A cluster (Kazuki et al., 2016). Moreover, it was demonstrated that auto-induced hepatic human P450s 3A4 and 3A5 (Murayama et al., 2014) can oxidize thalidomide to primary 5-hydroxy- and secondary dihydroxylated metabolites (Yamazaki et al., 2012; Chowdhury et al., 2014) in mice containing humanized liver cells (Nishiyama et al., 2015) to produce GSH adducts (Yamazaki et al., 2012) or protein adducts, including nonspecific modification of several protein networks (Yamazaki et al., 2016). In this auto-induction step, thalidomide and its human metabolite 5-hydroxythalidomide reportedly increased human hepatic P450 3A enzymes by direct activation of pregnane X receptor (PXR) (Murayama et al., 2014). The placenta is a key organ in fetal growth and development and controls maternal-to-fetal exchanges of nutrients and hormones by transport and metabolism processes (Prouillac and Lecoeur, 2010). These findings suggest the generation of reactive metabolites (Gordon et al., 1981) in the oxidation of thalidomide by human P450 enzymes both in livers and embryos, but there is no information available regarding placenta in terms of metabolic activation of thalidomide, in spite of some findings regarding nuclear receptor expressions in placenta (Geenes et al., 2011).
The commercially available BeWo placental cell line (Sarkar et al., 2006) has been recently used as an in vitro model for the placenta to study mechanisms of differentiation. However, there is no available information in terms of activities of drug metabolizing enzymes or xenobiotic transporters. During pregnancy, the function and expression levels of drug-metabolizing enzymes in the placenta generally change based on gestational stages or elevated plasma concentrations of steroidal hormones. Estrogens, cortisol, and progesterone reportedly modulate P450 catalytic function (Papageorgiou et al., 2013).
We report herein that analyses using three human placental preparations and placental BeWo cells revealed in vitro activation of thalidomide to 5-hydroxythalidomide with autoinduction of P450 3A enzymes in human placental BeWo cells in different culture medium systems.
MATERIALS AND METHODS
Human placenta and cells
Approval for the study using individual human placenta samples from normal childbirths was obtained from the Institutional Review Board of the Faculty of Medicine, Tottori University (Approval No. 1916). Placental BeWo cells (ATCC CCL-9; lot No. 59368210), obtained from American Type Culture Collection (Manassas, VA, USA), were cultured under recommended conditions with Ham’s F12K medium including 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA. In cases involving removal of endogenous hormones, 5% (v/v) charcoal-stripped fetal bovine serum (Thermo Fisher Scientific) was used. Induction of P450 activities of cultured BeWo cells were investigated with 20 µM rifampicin, 2000 µM phenobarbital, or 1000 µM thalidomide for 72 h according to the previous conditions used with human hepatocytes (Murayama et al., 2014).
P450-related mRNA levels and oxidation activities
BeWo cells (3 × 105 cells) were seeded in 24-well culture dishes. mRNA levels of human P450 3A4 (Hs01546612_m1), P450 3A5 (Hs00241417_m1), P450 2B6 (Hs04183483_g1), P450 3A7 (Hs00426361_m1), PXR (Hs1114267_m1 NR 1/2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs99999905_m1) were determined using a TaqMan system (Murayama et al., 2014), with total RNA fractions extracted using a Qiagen RNeasy Mini Kit (Hilden, Germany). The relative P450 mRNA levels were estimated after normalization in three independent amplifications. Drug oxidation activities were determined by HPLC-mass spectrometry as described previously (Murayama et al., 2014;Yajima et al., 2014).
RESULTS
Comparison of drug oxidation activities of human placental preparations and BeWo cells
In a preliminary study, placental preparations from one subject had detectable midazolam 1´-hydroxylation and thalidomide 5-hydroxylation activities, indicative of the potential for human P450 3A-dependent metabolic activation of thalidomide. In this study, placental microsomal fractions from three subjects showed detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities (Table 1). Human placental BeWo cells, cultured in the recommended media, also indicated detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. mRNA levels of P450 3A4 (expressed as ratios to GAPDH) in three placenta preparations (numbers 1, 2, and 3) and BeWo cells cultured in the recommend medium condition were 4 × 10−5, 1 × 10−5, 9 × 10−5, and 0.4 × 10−5 and those of P450 3A5/GAPDH were 1 × 10−4, 5 × 10−4, 9 × 10−4, and 4 × 10−4, respectively.
Table 1.
Drug oxidation activities of human placental microsomes and cultured BeWo cells.
| drug hydroxylation activities |
placental preparation | BeWo cells pmol product formed/h/106 cells |
||
|---|---|---|---|---|
|
| ||||
| 1 | 2 | 3 | ||
| pmol product formed/min/mg protein | ||||
|
| ||||
| bupropion | < 0.01 | < 0.01 | < 0.01 | 1.7± 0.1 |
| midazolam (1´) | 1.3 ± 0.3 | 1.3 ± 0.4 | 1.9 ± 0.5 | 40 ± 8 |
| midazolam (4) | 0.094 ± 0.030 | 0.073 ± 0.004 | 0.046 ± 0.006 | 2.1 ± 0.3 |
| thalidomide (5) | 0.024 ± 0.004 | 0.022 ± 0.003 | 0.020 ± 0.003 | 0.73 ± 0.18 |
Data are means and SD values of triplicate determinations. Placental microsomes (numbers 1, 2 and 3) and BeWo cells were incubated with 50 µM midazolam, 100 µM bupropion, or 500 µM thalidomide for 30 min in the presence of an NADPH-generating system and for 3 h in culture media containing 10% fetal bovine serum (v/v), respectively. The numbers in parentheses show the positions of hydroxylation of probe drugs.
Comparison of drug oxidation activities of BeWo cells in different culture system
The inducing effects of phenobarbital, rifampicin, and thalidomide on P450 3A4/5 and 2B6 in BeWo cells were investigated. Only rifampicin induced midazolam 1´-hydroxylation activity (Table 2), but mRNA levels of P450 3A4, 3A5, and 2B6 in BeWo cells were not significantly induced by these drugs under the present conditions (results not shown). To reduce any masking effects by endogenous hormones used in the recommended media, 10% fetal bovine serum (v/v) was replaced with 5% charcoal stripped fetal bovine serum (v/v) in the induction analysis. Thalidomide significantly induced the P450 3A4/3A5, 2B6 mRNA, and PXR levels 2- to 3-fold (Figure 1), but rifampicin only enhanced P450 3A5 and PXR mRNA under the modified media conditions. Under these modified conditions, thalidomide also significantly induced midazolam 1´-hydroxylation and thalidomide 5-hydroxylaion activities 3-fold but not bupropion hydroxylation activity (Table 3). Induction of drug oxidation activities by rifampicin was not seen in the modified medium condition, in spite of significant induction of midazolam 1´-hydroxylation activities mediated by P450 3A enzymes in BeWo cells cultured under the recommended medium conditions.
Table 2.
Induction of drug oxidation activities of cultured BeWo cells under the recommended conditions.
| drug hydroxylation |
untreated | treated with | ||
|---|---|---|---|---|
|
| ||||
| phenobarbital | rifampicin | thalidomide | ||
|
| ||||
| pmol product formed/h/106 cells | ||||
| bupropion | 2.6 ± 0.4 | 3.4 ± 0.7 | 3.2 ± 0.8 | 2.7 ± 0.5 |
| midazolam (1´) | 60 ± 23 | 52 ± 31 | 120 ± 21* | 47 ± 11 |
| midazolam (4) | 1.3 ± 0.3 | 1.7 ± 0.3 | 1.3 ± 0.3 | 1.9 ± 0.4 |
| thalidomide (5) | 0.43 ± 0.17 | 0.62 ± 0.25 | 0.63 ± 0.25 | 0.51 ± 0.37 |
Data are means and SD values of triplicate determinations. BeWo cells were cultured in the recommended media containing 10% fetal bovine serum (v/v) with 20 µM rifampicin, 2000 µM phenobarbital, or 1000 µM thalidomide for 72 h. The medium was replaced with a fresh medium containing 50 µM midazolam, 100 µM bupropion, or 500 µM thalidomide and further incubated for 3 h.
p < 0.05, one way ANOVA with Bonferroni post tests compared to untreated group.
Figure 1.
Induction of P450 3A4 (A), 3A5 (B), 2B6 (C), and PXR (D) mRNA levels in BeWo cells cultured in modified media containing 5% (v/v) charcoal-stripped fetal bovine serum by phenobarbital, rifampicin, and thalidomide. P450 inducers were added in the same manner as described in the legend for Table 3. *p < 0.05.
Table 3.
Induction of drug oxidation activities of cultured BeWo cells in the modified condition.
| drug hydroxylation |
untreated | treated with | ||
|---|---|---|---|---|
|
| ||||
| phenobarbital | rifampicin | thalidomide | ||
|
| ||||
| pmol product formed/h/106 cells | ||||
| bupropion | 2.9 ± 0.3 | 3.3 ± 0.8 | 2.3± 0.9 | 2.8 ± 0.7 |
| midazolam (1´) | 16 ± 9 | 32 ± 11 | 27 ± 10 | 60 ± 10* |
| midazolam (4) | 1.7 ± 0.2 | 1.6 ± 0.1 | 1.7 ± 0.1 | 1.7 ± 0.1 |
| thalidomide (5) | 0.23 ± 0.05 | 0.34 ± 0.09 | 0.33 ± 0.09 | 0.57 ± 0.18* |
Data are presented as means and SD values of triplicate determinations. BeWo cells were cultured in the modified media containing 5% (v/v) charcoal-stripped fetal bovine serum by phenobarbital, rifampicin, and thalidomide with 20 µM rifampicin, 2000 µM phenobarbital, or 1000 µM thalidomide for 72 h. Other details are as in the legend for Table 2.
p < 0.05, one way ANOVA with Bonferroni post tests compared to untreated group.
DISCUSSION
In this study, human placenta preparations and placental BeWo cells showed low but detectable and/or inducible P450 3A4/5 mRNA expression and drug oxidation activities. In a preliminary study, human placenta BeWo cells showed low but measurable drug oxidation activities (toward phenacetin, diclofenac, dextromethorphan, and alprazolam), in addition to bupropion, midazolam, and thalidomide (Table 1), in contrast to previous findings of only detectable P450 1A1/2-mediated activities in BeWo cells (Prouillac and Lecoeur, 2010). Thalidomide is an example of a human teratogen that is a substrate for human P450 enzymes (Hakkola et al., 1998) Thalidomide reportedly can act as an agonist for PXR in an assay for real-time co-regulator-nuclear receptor interaction and cause P450 Family 3A mRNA and enzyme induction in human hepatocytes and hepatic cell line HepaRG cells (Murayama et al., 2014). With regard to the PXR expression in placental BeWo cells, overlapping detection of other minor nuclear receptor subfamily 1 group I member 2 than PXR might be not ruled out in the current TaqMan assay system, but the similar high levels of PXR mRNA (Figure 1D) as well as those for P450 3A5 mRNA (Figure 1B) were seen in BeWo cells.
Regarding human fetal specific P450 3A7 in the placenta (Schuetz et al., 1993; Maezawa et al., 2010), mRNA levels of P450 3A7 have not been determined in the placental BeWo cells (Prouillac and Lecoeur, 2010). Because mRNA prepared from adult human livers showed no detectable P450 3A7 mRNA levels, we used the control samples from human fetal livers (Shimada, et al., 1996). Arbitrarily measured P450 3A7 mRNA/GAPDH levels in BeWo cells and fetal livers were 0.43 and 1.0, respectively, with commercial TaqMan systems. Consequently, the basal preparations for P450 mRNA determinations were different each other and therefore direct comparison of P450 3A4/5 and 3A7 was not achievable in this study. Under the present condition, P450 3A7 mRNA/GAPDH levels in normal placental preparations from three subjects (Table 1) were not detected (3A7 mRNA/GAPDH < 0.01). These normal placental preparations showed roughly similar activities of midazolam 1´- and 4-hydroxylation or thalidomide 5-hydroxylation (Table 1), presumably because these placental preparations had different levels of poorly detected P450 3A4 and extensively expressed P450 3A5 mRNA levels in combination.
It has been reported that human P450 3A4 induction in hepatic HepaRG cells could be consistently dependent on hydrocortisone hemisuccinate in the incubation medium supplied by the manufacturer (Papageorgiou et al., 2013). In that study, only cortisol of all the hormones tested individually induced human P450 3A4 expression and activity significantly in sandwich-cultured human hepatocytes (Papageorgiou et al., 2013). In the present study with BeWo cells, it could not be ruled out the possibility that the charcoal might remove any factors other than endogenous homomers from fetal bovine serum. However, in our preliminary experiments, interestingly, the resulting P450 3A induction ratios were high when hydrocortisone was omitted from basal culture medium and pregnancy-related hormones were added (unpublished results). The present results and previous findings collectively indicate that thalidomide is an inducer of human P450 3A enzymes in placenta as well as liver (but not a potent one), as judged by the catalytic activity and mRNA determinations in human placental cells cultured in the modified media. Similarly, weak induction of P450 2B6 by thalidomide was seen in BeWo cells in the present study in the similar manner as human hepatocytes (Murayama et al., 2014).
In conclusion, thalidomide may exert its variety of pharmacological and toxic actions through multiple mechanisms after bioactivation through its oxidation to 5-hydroxythalidomide and subsequent metabolic activation by human P450 enzymes autoinduced by thalidomide and its human primary metabolite 5-hydroxythalidomide in placenta and/or livers.
Acknowledgments
The authors thank Drs. Makiko Shimizu and Shotaro Uehara (Showa Pharmaceutical University) for their technical assistance and Drs. M. Oshimura and M. Osaki (Tottori University) for critical discussions. This work was supported in part by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 26460206 (H.Y.) and 15H04285 (Y.K.), Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.K.), and National Institutes of Health grant R01 GM118122 (F.P.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abbreviations
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- PXR
pregnane X receptor
References
- Chowdhury G, Shibata N, Yamazaki H, Guengerich FP. Human cytochrome P450 oxidation of 5-hydroxythalidomide and pomalidomide, an amino analog of thalidomide. Chem. Res. Toxicol. 2014;27:147–156. doi: 10.1021/tx4004215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geenes VL, Lim YH, Bowman N, Tailor H, Dixon PH, Chambers J, Brown L, Wyatt-Ashmead J, Bhakoo K, Williamson C. A placental phenotype for intrahepatic cholestasis of pregnancy. Placenta. 2011;32:1026–1032. doi: 10.1016/j.placenta.2011.09.006. [DOI] [PubMed] [Google Scholar]
- Gordon GB, Spielberg SP, Blake DA, Balasubramanian V. Thalidomide teratogenesis: evidence for a toxic arene oxide metabolite. Proc. Natl. Acad. Sci. U.S. A. 1981;78:2545–2548. doi: 10.1073/pnas.78.4.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hakkola J, Pelkonen O, Pasanen M, Raunio H. Xenobiotic-metabolizing cytochrome P450 enzymes in the human feto-placental unit: role in intrauterine toxicity. Crit. Rev. Toxicol. 1998;28:35–72. doi: 10.1080/10408449891344173. [DOI] [PubMed] [Google Scholar]
- Kazuki Y, Akita M, Kobayashi K, Osaki M, Satoh D, Abe S, Takehara S, Kazuki K, Yamazaki H, Kamataki T, Oshimura M. Thalidomide-induced limb abnormalities in a humanized CYP3A mouse model. Sci. Rep. 2016;6:21419. doi: 10.1038/srep21419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maezawa K, Matsunaga T, Takezawa T, Kanai M, Ohira S, Ohmori S. Cytochrome P450 3As gene expression and testosterone 6β-hydroxylase activity in human fetal membranes and placenta at full term. Biol. Pharm. Bull. 2010;33:249–254. doi: 10.1248/bpb.33.249. [DOI] [PubMed] [Google Scholar]
- Murayama N, van Beuningen R, Suemizu H, Guguen-Guillouzo C, Shibata N, Yajima K, Utoh M, Shimizu M, Chesne C, Nakamura M, Guengerich FP, Houtman R, Yamazaki H. Thalidomide increases human hepatic cytochrome P450 3A enzymes by direct activation of pregnane X receptor. Chem. Res. Toxicol. 2014;27:304–308. doi: 10.1021/tx4004374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishiyama S, Suemizu H, Shibata N, Guengerich FP, Yamazaki H. Simulation of human plasma concentrations of thalidomide and primary 5-hydroxylated metabolites explored with pharmacokinetic data in humanized TK-NOG mice. Chem. Res. Toxicol. 2015;28:2088–2090. doi: 10.1021/acs.chemrestox.5b00381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Papageorgiou I, Grepper S, Unadkat JD. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines. Drug Metab. Dispos. 2013;41:281–290. doi: 10.1124/dmd.112.049015. [DOI] [PubMed] [Google Scholar]
- Prouillac C, Lecoeur S. The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies. Drug Metab. Dispos. 2010;38:1623–1635. doi: 10.1124/dmd.110.033571. [DOI] [PubMed] [Google Scholar]
- Sarkar P, Shiizaki K, Yonemoto J, Sone H. Activation of telomerase in BeWo cells by estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin in co-operation with c-Myc. Int. J. Oncol. 2006;28:43–51. [PubMed] [Google Scholar]
- Schuetz JD, Kauma S, Guzelian PS. Identification of the fetal liver cytochrome CYP3A7 in human endometrium and placenta. J. Clin. Invest. 1993;92:1018–1024. doi: 10.1172/JCI116607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimada T, Yamazaki H, Mimura M, Wakamiya N, Guengerich FP, Inui Y. Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal livers and adult lungs. Drug Metab. Dispos. 1996;24:515–522. [PubMed] [Google Scholar]
- Yajima K, Uno Y, Murayama N, Uehara S, Shimizu M, Nakamura C, Iwasaki K, Utoh M, Yamazaki H. Evaluation of 23 lots of commercially available cryopreserved hepatocytes for induction assays of human cytochromes P450. Drug Metab. Dispos. 2014;42:867–871. doi: 10.1124/dmd.113.056804. [DOI] [PubMed] [Google Scholar]
- Yamazaki H, Suemizu H, Kazuki Y, Oofusa K, Kuribayashi S, Shimizu M, Ninomiya S, Horie T, Shibata N, Guengerich FP. Assessment of protein binding of 5-hydroxythalidomide bioactivated in humanized mice with human P450 3A-chromosome or hepatocytes by two-dimensional electrophoresis/accelerator mass spectrometry. Chem. Res. Toxicol. 2016;29:1279–1281. doi: 10.1021/acs.chemrestox.6b00210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamazaki H, Suemizu H, Shimizu M, Igaya S, Shibata N, Nakamura N, Chowdhury G, Guengerich FP. In vivo formation of dihydroxylated and glutathione conjugate metabolites derived from thalidomide and 5-hydroxythalidomide in humanized TK-NOG mice. Chem. Res. Toxicol. 2012;25:274–276. doi: 10.1021/tx300009j. [DOI] [PMC free article] [PubMed] [Google Scholar]