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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 5  |  Page : 506-512  

Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells


1 Division of Pre-clinic, Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand
2 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
3 Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
4 Center for Pathophysiology, Infectiology and Immunology, Institute for Pathophysiology and Allergy Research, Medical University of Vienna, 1090 Vienna, Austria

Date of Submission27-Apr-2020
Date of Decision20-May-2020
Date of Acceptance14-Sep-2020
Date of Web Publication30-Nov-2020

Correspondence Address:
Isabella Ellinger
Center for Pathophysiology, Infectiology and Immunology, Institute for Pathophysiology and Allergy Research, Medical University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna
Austria
Kanokwan Jarukamjorn
Faculty of Pharmaceutical Sciences, Khon Kaen University, 123 Mitraparb Road, Khon Kaen 40002
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_164_20

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   Abstract 


Background: The human placenta metabolizes many endogenous substances, drugs, and xenobiotics. Cytochrome P450 family 1 (CYP1) is expressed in both early- and full-term placenta. The Thai medicinal plant Pueraria candollei var. mirifica (PM) is traditionally consumed for rejuvenation and has neuroprotective, anti-osteoporotic, and antioxidant activities. Objectives: The objective of this study was to compare the effects of PM and the CYP1A inducer β-naphthoflavone (BNF) on the expression of CYP1, aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and the transporter ABCG2. Materials and Methods: Human choriocarcinoma BeWo cells were treated with BNF (10 μM), ethanolic extract of PM (PM-EtOH), or column chromatographic fractions of PM-EtOH (F2, F4, and F6) at 1, 10, and 100 μg/mL for 24 h. The mRNA expression of target genes was determined using real-time quantitative polymerase chain reaction. The activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP1, was measured at the RNA harvesting time point. Results and Discussion: PM-EtOH, F2, and F4 significantly induced EROD activity and expression of CYP1A1 and CYP1A2 while CYP1B1 and AHR were slightly suppressed and ARNT was unchanged. ABCG2 was slightly induced by F2. Therefore, the expression of CYP1 in BeWo cells appears to be independent of the AHR/ARNT regulatory pathway. Conclusion: The use of PM-containing products at high quantities or for long periods during pregnancy is of concern due to likely herb–drug interactions and toxicological risks through activation of CYP1A1 and CYP1A2 transcription.

Keywords: Cytochrome P450, ethoxyresorufin-O-deethylase, nuclear receptor, placenta, transporter


How to cite this article:
Chatuphonprasert W, Kitisripanya T, Putalun W, Ellinger I, Jarukamjorn K. Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells. Phcog Mag 2020;16, Suppl S2:506-12

How to cite this URL:
Chatuphonprasert W, Kitisripanya T, Putalun W, Ellinger I, Jarukamjorn K. Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells. Phcog Mag [serial online] 2020 [cited 2021 Jan 25];16, Suppl S2:506-12. Available from: http://www.phcog.com/text.asp?2020/16/5/506/301876



SUMMARY

  • The induction of CYP1A1 and CYP1A2 expression by the ethanolic extract of Pueraria candollei var. mirifica and its chromatographic fractions in BeWo cells are of concerns for risks of carcinogenesis and drug interactions, especially a use at high amount or in pregnancy.




Abbreviations Used: AHR: aryl hydrocarbon receptor; ARNT: AHR nuclear translocator; BNF: β-naphthoflavone; CYP: Cytochrome P450; EROD: ethoxyresorufin-O-deethylase; PM: Pueraria candollei var. mirifica; PM-EtOH: ethanolic extract of Pueraria candollei var. mirifica.


   Introduction Top


Pueraria candollei var. mirifica (PM) is a Thai medicinal herb with several pharmacological activities, for example, antioxidation,[1],[2] osteoporosis prevention,[3],[4] and neuroprotection,[5] as well as anti-elastase and anti-collagenase activities useful for skincare products.[6] The human placenta produces the hormones and proteins necessary to maintain pregnancy and to support normal fetal development. In addition, the placenta metabolizes many exogenous substances, including drugs, environmental pollutants, and xenobiotics.[7] Cytochrome P450 family 1 (CYP1) comprises the most common metabolizing enzymes for xenobiotics and toxicants, and CYP1 enzymes are expressed in the placenta. The CYP1 family consists of three isoforms CYP1A1, CYP1A2, and CYP1B1.[8] In the human choriocarcinoma BeWo cell line, CYP1A1 and CYP1A2 are expressed and inducible.[9] CYP1B1 is commonly found in extrahepatic tissues such as lungs, lymphocytes, mammary glands, and placenta.[10],[11] Polycyclic aromatic hydrocarbons (PAHs), such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC), benzo[a]pyrene (B[a]P), or β-naphthoflavone (BNF), are broadly distributed environmental contaminants that cause several toxicities in mammals, including carcinogenesis, teratogenesis, and immune dysfunction.[12] PAHs induce the expression of various metabolizing enzymes through activation of the aryl hydrocarbon receptor (AHR), which forms a dimer with the aryl hydrocarbon receptor nuclear translocator (ARNT) that binds to the aryl hydrocarbon response element upstream of the CYP1 genes.[13] Breast cancer resistance protein (BCRP/MXR/ABCG2) is a member of the ATP-binding cassette (ABC) transporter superfamily that has been associated with multidrug resistance in many diseases.[14] ABCG2 is highly expressed in several human tissues, for example, colon, liver, small intestine, and placenta, and contributes to the protective function of those tissues.[14] In human placenta, ABCG2 is most abundantly expressed in the apical membrane of the placental syncytiotrophoblast[15] where it mediates the unidirectional transport of substrates, including PAHs, as an efflux pump.[16]

In experiments conducted in silico and in ICR mouse microsomes, five PM phytoestrogens (miroestrol, kwakhurin, deoxymiroestrol, methoxyisomiroestrol, and isomiroestrol) were shown to inhibit the activities of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) enzymes, which act as markers for CYP1 activities.[17] Similarly, PM decreased the activities of benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase, which represent CYP2B activity, in both regular diet- and high-cholesterol diet-fed male Wistar rats.[18] Miroestrol and deoxymiroestrol increased uterus weight and volume in female C57BL/6 mice[19] accompanied by induction of Cyp2b9 expression and suppression of Cyp1a2 expression in mouse hepatocytes.[20] To date, information about the effect of PM on the expression of CYP1 and ABCG2 in placenta models is limited. Therefore, the present study aimed to examine the effects of an ethanolic extract of PM on the expression of CYP1 (CYP1A1, CYP1A2, and CYP1B1), the CYP upstream regulators, AHR and ARNT, and the transporter ABCG2 in human choriocarcinoma BeWo cells.


   Materials and Methods Top


Chemicals and reagents

Authentic standards of isoflavonoids and chromenes were kindly provided by Prof. Waraporn Putalun (Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand). Dulbecco's modified Eagle's medium (DMEM) and cell culture supplements were supplied by Gibco® (Thermo Fisher Scientific, MD, USA). Ethoxyresorufin, resorufin standard, and resazurin were supplied by Sigma-Aldrich Chemical (St. Louis, MO, USA). Reverse transcription reagents, TaqMan™ probe and primers, and TaqMan™ gene expression assays were products of Applied Biosystems (California, USA). All other laboratory reagents and equipment were of the highest purity and standard quality from commercial suppliers.

The Pueraria candollei var. mirifica crude extract and the column chromatographic fraction preparation PM tuberous roots (Reference specimen NI-PSKKU 007-010) were previously collected in Ubon Ratchathani, Thailand.[21] The dried powder of PM was extracted in absolute ethanol using a Soxhlet apparatus at 60°C for 3 h. The PM ethanolic extract (PM-EtOH) was filtered and evaporated at 60°C (2.26% yield). Then, the PM-EtOH extract was fractionated on a Silica Gel 60 column with hexane/ethanol mixtures of increasing polarity (from 0% to 100% of ethanol; 50 mL/h). A 5 mL aliquot of each fraction was analyzed by thin-layer chromatography (TLC), and the fractions with identical TLC patterns were pooled to obtain the F2, F4, and F6 fractions, which were subsequently evaporated. A 1 mg aliquot of PM-EtOH, F2, F4, and F6 was dissolved in 1 mL ethanol and filtered through a 0.2 μm-membrane for high-performance liquid chromatographic (HPLC) analysis.

High-performance liquid chromatographic condition

The HPLC was performed using a PerkinElmer Series 200 LC pump coupled with a reversed-phase C18 column (LiChroCART®, 125 mm × 4 mm, 5 μm particle size) and a PerkinElmer 785A UV/VIS detector. The detection was set at a wavelength of 280 nm. The flow rate was set at l mL/min. A 20 μL aliquot of each sample was subjected to HPLC. The gradient elution was programmed using a dual pumping system by varying the proportion of solvent A (1% acetic acid in water) and solvent B (100% acetonitrile) with a linear gradient program of 10%–15% B over 0–15 min, 15%–20% B over 15–35 min, 20%–35% B over 35–40 min, 35%–45% B over 40–50 min, 45%–80% B over 50–55 min, and finally maintained at 80% B for 5 min to elute any unwanted matrix. Then, the gradient elution program was set to 80%–10% B over 60–70 min and returned to the initial conditions.[22] A series of standard solutions was prepared by mixing the standards of isoflavonoids and chromenes to obtain the solutions with final concentrations of each isoflavonoid (puerarin, daidzin, genistin, daidzein, genistein, and kwakhurin) at 6.25 μg/mL and each chromene (isomiroestrol, miroestrol, and deoxymiroestrol) at 12.5 μg/mL.

Cell culture

BeWo cell clone b24 was cultured in high-glucose DMEM with 10% fetal calf serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.[23] BeWo cells (5 × 105 cells/well) were seeded in a 6-well plate for 24 h before treatment. The cells (n = 4–5 wells) were incubated with 0.2% dimethyl sulfoxide (DMSO, control), 10 μM BNF, or 1, 10, and 100 μg/mL of PM-EtOH, F2, F4, or F6 in phenol red-free medium for further 24 h.

Cell viability assessment

After treatment for 24 h, resazurin was added to the medium at a final concentration of 1 μM and incubated for 1 h at 37°C. The fluorescence intensity was measured at 530/590 nm excitation/emission. The percentage of cell viability was calculated compared to the control (0.2% DMSO).[24]

Ethoxyresorufin-O-deethylase activity assay

The EROD activity assay was modified from a previous report.[25] Ethoxyresorufin at the final concentration of 5 μM was incubated with the cells at 37°C for 3 h. Formation of resorufin in the medium was analyzed using spectrofluorometry at 530/590 nm excitation/emission and compared with a resorufin standard (10–200 nM). EROD activity was calculated as formation of resorufin (fmole) per min.

Total RNA isolation, cDNA synthesis, and real-time polymerase chain reaction determination

Total RNA was isolated using the peqGOLD TriFast™ Kit (Peqlab Biotechnologie GmbH, Erlangen, Germany). A 1 μg aliquot of total RNA was used to examine RNA integrity by 1.2% agarose gel electrophoresis in 1× Tris-borate-EDTA (TBE) buffer containing GelRed™ (Biotium, Koln, Germany) at 120 V for 20 min. Total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Life Technologies, Carlsbad, USA). Nontemplate control was a negative control using RNAse/DNAse-free water instead of any RNA sample. Thermal cycler was set to generate cDNA at a multiphase condition: 25°C (10 min), 37°C (120 min), 85°C (5 min), and 4°C (∞). For quantitative polymerase chain reaction (qPCR), the 7900HT Fast Real-Time PCR System and TaqMan™ Gene Expression Master Mix (Applied Biosystems, California, USA) in combination with the following TaqMan™ probes and primers of the following genes: CYP1A1 (Hs01054797_g1), CYP1A2 (Hs00167927_m1), CYP1B1 (Hs02382916_s1), AHR (Hs00907314_m1), ARNT (Hs01121918_m1), ABCG2 (Hs01053790_m1), and two reference genes, ubiquitin (UBC; Hs00824723_m1) and TATA box-binding protein (TBP; Hs99999910_m1), were used. The changes in Ct (ΔCt) between the genes of interest and the geometric mean of the housekeeping genes and the changes in ΔCt between the treatments and the controls (ΔΔCt) were calculated. The fold change in expression between samples was calculated from 2-(ΔΔCt).[26]

Statistical analysis

From the qPCR experiment, the average fold differences from each group (n = 4–5 per group) were plotted and analyzed using one-way analysis of variance followed by Tukey's post hoc test (SPSS version 23, Chicago, IL, USA). P ≤ 0.05 was considered as statistically significant.


   Results Top


Quantitative analysis of isoflavonoids and chromenes in Pueraria candollei var. mirifica ethanolic extract and its column chromatographic fractions, F2, F4, and F6

An HPLC profile of pure isoflavonoids and chromenes is depicted in [Figure 1]. PM-EtOH (1 mg/mL) contained puerarin (71.53 ± 0.81 μg/mL), daidzin (3.01 ± 0.05 μg/mL), isomiroestrol (12.54 ± 0.52 μg/mL), daidzein (0.98 ± 0.02 μg/mL), and kwakhurin (4.82 ± 0.04 μg/mL). F2 contained daidzein (4.37 ± 0.01 μg/mL) and kwakhurin (25.94 ± 0.03 μg/mL). F4 had an HPLC profile similar to PM-EtOH and contained puerarin (116.98 ± 5.67 μg/mL), daidzin (5.43 ± 0.20 μg/mL), isomiroestrol (10.94 ± 0.24 μg/mL), daidzein (2.16 ± 0.02 μg/mL), and kwakhurin (5.70 ± 0.13 μg/m). F6 contained puerarin (86.47 ± 3.63 μg/mL) and daidzin (2.77 ± 0.05 μg/mL).
Figure 1: High-performance liquid chromatographic chromatogram of authentic standard isoflavonoids and chromenes, Pueraria candollei var. mirifica ethanolic extract, and its column chromatographic fractions F2, F4, and F6. Authentic standard isoflavonoids (puerarin, daidzin, genistin, daidzein, genistein, and kwakhurin) at 6.25 μg/mL and authentic standard chromenes (isomiroestrol, miroestrol, and deoxymiroestrol) at 12.5 μg/mL (blue line); Pueraria candollei var. mirifica ethanolic extract at 1 mg/mL (magenta line); the column chromatographic fraction number 2 of the Pueraria candollei var. mirifica ethanolic extract (F2) at 1 mg/mL (green line); the column chromatographic fraction number 4 of the Pueraria candollei var. mirifica ethanolic extract (F4) at 1 mg/mL (red line); (e) the column chromatographic fraction number 6 of the Pueraria candollei var. mirifica ethanolic extract (F6) at 1 mg/mL (gray line)

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Effects of Pueraria candollei var. mirifica ethanolic extract and its column chromatographic fractions on ethoxyresorufin-O-deethylase activity

The CYP1A inducer BNF (10 μM) significantly induced EROD activity in BeWo cells [Figure 2]. PM-EtOH (at 10 and 100 μg/mL), F2 (at 1 and 10 μg/mL), and F4 (at 1, 10, and 100 μg/mL) extensively increased EROD activity. Likewise, F6 at the highest concentration of 100 μg/mL significantly elevated EROD activity. F2 was cytotoxic to BeWo cells at concentrations higher than 50 μg/mL, with <80% cell viability after treatment (data not shown), while other groups had >80% cell viability. Hence, only 1 and 10 μg/mL of F2 were further examined.
Figure 2: Effects of Pueraria candollei var. mirifica ethanolic extract and its column chromatographic fractions F2, F4, and F6 on ethoxyresorufin-O-deethylase activity. DMSO, 0.2% dimethyl sulfoxide; BNF, 10 μM β-naphthoflavone; PM-EtOH, Pueraria candollei var. mirifica ethanolic extract at 1, 10, and 100 μg/mL; F2, F4, F6, the column chromatographic fraction number 2, 4, and 6 of the Pueraria candollei var. mirifica ethanolic extract at 1, 10, and/or 100 μg/mL. *P < 0.05, **P < 0.001 versus dimethyl sulfoxide;#P < 0.05,##P < 0.001 versus BNF

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Effects of Pueraria candollei var. mirifica ethanolic extract and its column chromatographic fractions on the expression of CYP1A1, CYP1A2, CYP1B1, and ABCG2 mRNAs

BNF (10 μM) significantly induced the expression of CYP1A1 [143-fold, [Figure 3]a and CYP1A2 [10.7-fold, [Figure 3]b mRNAs in BeWo cells. PM-EtOH, F2, and F4 extensively induced the expression of CYP1A1 mRNA (47–215-fold) in a concentration-dependent manner, while F6 did not alter CYP1A1 expression [Figure 3]a. Similarly, PM-EtOH, F2, and F4 significantly induced the expression of CYP1A2 mRNA (3–7-fold), while F6 did not [Figure 3]b. In contrast, the expression of CYP1B1 mRNA was suppressed by BNF, PM-EtOH, F2, and the lowest concentration of F6 (1 μg/mL) [Figure 3]c. The expression of ABCG2 mRNA was significantly increased by F2 at a concentration of 10 μg/mL [Figure 3]d, while all other treatments did not modify ABCG2 expression.
Figure 3: Effects of PM-EtOH and its column chromatographic fractions F2, F4, and F6 on the gene expression of CYP1A1, CYP1A2, CYP1B1, and ABCG2. Fold change in mRNA expression of the target genes, i.e., CYP1A1 (a), CYP1A2 (b), CYP1B1 (c), and ABCG2 (d), normalized to the reference genes TATA box-binding protein (TBP) and ubiquitin C (UBC). DMSO, 0.2% dimethyl sulfoxide; BNF, 10 μM β-naphthoflavone; PM-EtOH, Pueraria candollei var. mirifica ethanolic extract at 1, 10, and 100 μg/mL; F2, F4, F6, the column chromatographic fraction number 2, 4, and 6 of the PM-EtOH at 1, 10, and/or 100 μg/mL. *P < 0.05, **P < 0.001 versus dimethyl sulfoxide;#P < 0.05,##P < 0.001 versus BNF;$P < 0.05

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Effects of Pueraria candollei var. mirifica ethanolic extract and its column chromatographic fractions on the expression of AHR and ARNT

The expression of AHR mRNA was significantly suppressed by PM-EtOH (10 and 100 μg/mL) and F6 (all concentrations) [Figure 4]a. No significant changes were observed for the expression of ARNT mRNA following any treatment [Figure 4]b.
Figure 4: Effects of PM-EtOH and its column chromatographic fractions F2, F4, and F6 on the gene expression of aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT). Fold change in mRNA expression of the target genes, i.e., AHR (a) and ARNT (b), normalized to the reference genes TATA box-binding protein (TBP) and ubiquitin C (UBC). DMSO, 0.2% dimethyl sulfoxide; BNF, 10 μM β-naphthoflavone; PM-EtOH, Pueraria candollei var. mirifica ethanolic extract at 1, 10, and 100 μg/mL; F2, F4, F6, the column chromatographic fraction number 2, 4, and 6 of the PM-EtOH at 1, 10, and/or 100 μg/mL. *P < 0.05 versus dimethyl sulfoxide

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   Discussion Top


Two chromenes, miroestrol and deoxymiroestrol, were not found in the PM-EtOH extract, presumably because they were lost during Soxhlet extraction, which was employed instead of maceration.[22],[27] Miroestrol and deoxymiroestrol possess potent estrogenic effects,[28] and these two compounds should be avoided in pregnancy. In this study, the Soxhlet extraction yielded the isoflavonoid, puerarin, which has shown nephro- and retinoprotective activities in diabetic models.[29],[30] In MCF-7 cells, an ethanolic extract of PM caused proliferation at 1 μg/mL and was antiproliferative at 100 μg/mL.[31] In this study, we chose the concentrations of the PM extracts (1, 10, and 100 μg/mL) to ensure >80% cell viability and prevent the inhibition of normal processes in cells. We determined CYP1A1 mRNA expression at 24 h as a previous study that B[a]P induced CYP1A1 in BeWo cells continuously from 8 to 96 h, reaching a maximum level of induction at 24 h.[32]

PAHs are ubiquitous environmental pollutants present wherever organic material is burned, for example, cigarette smoke, automobile exhaust, cooked foods, wood smoke, and a number of environmental products, such as coal, tar, petroleum, and cutting fluid.[33] Most PAHs are inducers of CYP1 (i.e., CYP1A1, CYP1A2, and CYP1B1), which is mainly responsible for formation of their toxic metabolites.[34],[35] Such activation may also occur at the placental level, and PAHs can induce placental enzymes including CYP1.[8] Here, we chose BNF, a PAH that has been used as a CYP1 inducer in several models.[36],[37],[38]

Increased CYP1A1 activity in placenta of female smokers has been associated with pregnancy complications, such as premature birth, intrauterine growth retardation, structural abnormalities, fetal death or placental abruption, and a risk of low birth weight, low birth length, and low head circumference.[39],[40] In smokers, a significant increase in the levels of CYP1A1 and CYP1B1 transcripts has been observed.[41] EROD activity has been widely used as a marker for CYP1 activity.[42],[43] EROD activity in BeWo cells has been shown to be sensitive to inducers, such as 3MC, 1,2-benzanthracene, and BNF (10–50 μM).[9] However, in this study, EROD activity [Figure 2] was not relative to the expression of CYP1B1 mRNA [Figure 3]c. There was a report on different regulations of CYP1A1 and CYP1B1 expression in human medulloblastoma cells (UW228-3),[44] and in another choriocarcinoma cell line JEG3, TCDD induced the expression of CYP1A1 and AHR mRNAs but not those of CYP1B1 and ARNT.[8],[45] Therefore, regulatory pathways of CYP1A1 and CYP1B1 in BeWo cells might be differently affected by xenobiotics. Moreover, EROD activity did not appear to be an appropriate enzymatic marker for CYP1B1 activity. The combination of dexamethasone and BNF additionally increased EROD activity,[9] and the transcriptional regulation of AHR-mediated pathways through dexamethasone induction has been reported.[46],[47] However, another report demonstrated that cell-specific induction of CYP1 was not related to the expression of AHR, ARNT, or estrogen receptors (ERs) in multiple human cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma).[34] In BeWo cells, the expression of AHR and ARNT mRNAs did not correlate with either CYP1 expression or EROD activity. This is in contrast to placenta, where CYP1A1 is transcriptionally upregulated through AHR.[40] Several studies have reported reactive oxygen species (ROS) production by PAH activating Ahr and inducing Cyp1 expression, especially in mouse livers.[48],[49],[50] However, the expression of AHR and ARNT mRNAs did not correlate with CYP1 expression in this study. Whether AhR-independent CYP1A activation is linked to ROS activation in BeWo cells remains to be demonstrated. Additional mechanisms involving transcription factors such as ER, androgen receptor, or nuclear factor-κB (NF-κB) could mediate the biological effects of AHR on CYP1.[51] Differential effects of PAHs have also been observed in JEG-3 and BeWo placental cells.[52] AHR-NF-κB interaction and AHR-ER cross-talk caused the inhibition of CYP1A1 and CYP1B1 proteins in BeWo cells, while CYP1A1 induction was not associated with NF-κB in JEG-3 cells.[52] Therefore, further examination of the other nuclear receptor-mediated pathways that are involved in the regulation of CYP1 transcription in BeWo cells is required.

PAHs exposure has also been associated with epigenetic alterations, including cytosine methylation, genome instability, and subsequent cancer risk.[53] Hence, the induction of CYP1A1 and CYP1A2 and increased EROD activity caused by PM-EtOH and its fractions should be a concern to pregnant women exposed to this plant due to the increased risk to the fetus. Regarding the HPLC results [Figure 1], F6 (the fraction containing puerarin and daidzin) showed less potential to induce the expression of CYP1A1 and CYP1A2 mRNAs compared to F2 and F4. However, PM-EtOH and F6 both suppressed the expression of AHR mRNA, which is involved in many critical processes during pregnancy, including fetal development.[54]

We previously reported the inhibitory effects of PM on EROD and MROD activities in mouse liver microsomes,[17] anin vitro study that has a limitation because it can only reveal inhibitory effects. In rat livers, PM did not affect CYP1A1 and CYP3A but did suppress CYP1A2, CYP2B1/2B2, and CYP2E1.[18]ABCG2, also known as the BCRP, is highly expressed in placental syncytiotrophoblasts at the apical membrane.[15] To date, the physiological role of ABCG2 in the placenta is not clear. Recent data suggest that ABCG2 plays a key role in protecting the fetus against toxic xenobiotics, endogenous substances, drugs, and metabolites by efflux pumping them across the placental barrier.[15] Some flavonoids (e.g., silymarin, hesperetin, quercetin, and daidzein) increased the activity of BCRP/ABCG2 in BCRP-overexpressing cell lines.[55] Miroestrol and deoxymiroestrol, the two potent chromene phytoestrogens in PM, suppressed the expression of BSEP (bile salt export pump, ABCB11) and MRP2 (multidrug resistance protein, ABCC2) mRNAs in mouse livers.[4] Information about the effect of PM and its constituents on other transporters is still limited. In this study, although miroestrol and deoxymiroestrol were not found in PM-EtOH and its fractions, F2 did induce ABCG2 expression, which might increase the efflux of xenobiotic(s) from fetus to mother.


   Conclusion Top


PM-EtOH, F2, and F4, which contained puerarin, daidzin, isomiroestrol, daidzein, and kwakhurin, significantly induced EROD activity, corresponding to an increase in the expression of CYP1A1 and CYP1A2 mRNAs in BeWo cells. However, the induction of CYP1A was not dependent on AHR/ARNT. These findings suggest that the use of PM during pregnancy carries a risk of modified placental CYP1A activity.

Acknowledgements

WC sincerely thanks the Faculty of Pharmaceutical Sciences, Khon Kaen University, and Faculty of Medicine, Mahasarakham University, Thailand. The authors thank Dr. Glenn Borlace, Khon Kaen University, for English language assistance.

Financial support and sponsorship

Austrian South-East Asian University Partnership Network for Ernst Mach Grant to work at the Medical University of Vienna, Austria, and the Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (Grant No. PANPB2561),

Conflicts of interest

There are no conflicts of interest.



 
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