The influence of soybean extract on the expression level of selected drug transporters, transcription factors and cytochrome P450 genes encoding phase I drug-metabolizing enzymes

The influence of soybean extract on the

expression level of selected drug transporters, transcription factors and cytochrome P450 genes encoding phase I drug-metabolizing enzymes

Wpływ ekstraktu sojowego na poziom ekspresji wybranych transporterów leków, czynników transkrypcyjnych i genów cytochromu P450 kodujących enzymy I fazy metabolizmu leków

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1 Laboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences, Poznan, Poland

2 Department of Quality Control of Medicinal Products and Dietary Supplements, Institute of Natural Fibres and Medicinal Plants, Poznan, Poland

3 Department of Pharmacology, University of Medical Sciences, Poznan, Poland

4 Department of Pharmacology and Experimental Biology, Institute of Natural Fibers and Medicinal Plants, Poznan, Poland

5 Department of Gastroenterology and Metabolic Diseases, Medical University of Warsaw, Poland

6 Division of Gynecology and Obstetrics, Podhale Multidisciplinary Hospital, Nowy Targ, Poland

7 Division of Perinatology and Women’s Diseases, Poznan University of Medical Sciences, Poznan, Poland

8 Department of General Pharmacology and Pharmacoeconomics, Pomeranian Medical University, Szczecin, Poland

Abstract

Objective: Soybean phytoestrogens, such as genistein and daidzein, reduce climacteric symptoms and the risk of certain chronic diseases such as cancer and cardiovascular diseases. Despite their widespread use in functional foods and dietary supplements, there is very little data available on their safety and herb-drug interactions, espe- cially with antineoplastic agents. Hence, the aim of our study was to assess the effects of soybean extracts on the expression level of CYP genes and their transcriptional factors. We also investigated the effect of soybean on the mRNA level of transporters, such as P-glycoprotein (MDR1) and multidrug resistance-associated proteins (MRP1, MRP2).

Materials and methods: Wistar rats were fed a standardized soybean extract (100 mg/kg, p.o.). cDNA was syn- thesized from total RNA isolated from different tissues (liver and intestinal epithelium) using reverse transcription.

Gene expression level was analyzed by RT-PCR method.

Results: We demonstrated a significant increase of CYP1A1 mRNA level (by 89%, p=0.002 and 125%, p=0.004) as compared with the control group. An increase of AHR and CAR expression after 10 days was also observed (by 60%, p<0.001 and 52%, p>0.05, respectively).

Otrzymano: 11.12.2014

Zaakceptowano do druku: 30.01.2014 Adres do korespondencji:

Anna Bogacz

Laboratory of Experimental Pharmacogenetics Department of Clinical Pharmacy and Biopharmacy Poznan University of Medical Sciences, Poznan, Poland 14 Św. Marii Magdaleny, 60-861 Poznań

e-mail: aniabogacz23@o2.pl

Introduction

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Additionally, an inductive effect for CYP2D1 by 22% (p=0.008), Mdr1a by 267% (p<0.0001), Mdr1b by 86%

(p<0.0001), Mrp1 by 9-fold (p<0.0001), Mrp2 by 83% (p<0.0001) in the liver and for Mrp2 by 35% (p<0.001) in the intestinal epithelium, was evaluated. A significant decrease of mRNA level was observed for CYP3A1 (human CY- P3A4) in the liver and Mdr1b in the intestinal epithelium. Moreover, we also showed a slight decrease in the amount of mRNA for CAR, PXR and ARNT after 3 days.

Conclusions: Our results suggest that Glycine max may change the expression level of CYPs, especially CYP3A4 and CYP1A1, involved in biotransformation of xenobiotics (drugs, procarcinogens) and may participate in clinically significant interactions with drugs metabolized by these enzymes. Moreover, an increase of CYP1A1 (homologue to human CYP1A1) mRNA level may not only reduce the carcinogenicity of foreign compounds, but may also ac- tivate some compounds to their carcinogenicity. In case of transporters, it is considered that an increase of their expression in the body may lead to increased fetoprotection. Also, it may reduce both, the exposure of sensitive tissues (e.g. brain, placenta) to xenobiotics and treatment effectiveness of certain diseases. Hence, the search for a safe substance that could effectively modulate transporter activity, especially in the treatment of certain hormone -dependent disorders. e.g. osteoporosis and breast cancer, occurring mainly in postmenopausal period, continues.

Key words: CYP enzymes / transporters / soybean / interactions / / transcriptional factors / expression level /

Streszczenie

Cel pracy: Fitoestrogeny sojowe, takie jak genisteina i daidzeina redukują symptomy menopauzy i zmniejszają ryzyko wystąpienia niektórych chorób w tym nowotworów i chorób serca. Pomimo szerokiego zastosowania izo- flawonów pod postacią funkcjonalnej żywności i suplementów diety, dane dotyczące bezpieczeństwa jak również interakcji typu preparat roślinny-lek syntetyczny, szczególnie uwzględniając leki przeciwnowotworowe są bardzo ograniczone. Celem badania była ocena wpływu ekstraktu sojowego na poziom ekspresji genów CYP i ich czyn- ników transkrypcyjnych. Zbadano również wpływ soi na poziom mRNA transporterów obejmując glikoproteinę P (MDR1) i białka związane z opornością wielolekową (MRP1, MRP2).

Materiał i metody: Szczury rasy Wistar traktowano ekstraktem sojowym (100 mg/kg, p.o.). cDNA syntetyzowano z całkowitego RNA z tkanki wątrobowej i nabłonka jelita stosując odwrotną transkrypcję. Poziom ekspresji genów analizowano wykorzystując metodę RT-PCR.

Wyniki: Wykazano znaczny wzrost poziomu mRNA CYP1A1 o 89% (p=0,002) i 125% (p=0,004) w porównaniu do grupy kontrolnej. Wzrost ekspresji AHR i CAR był również obserwowany odpowiednio o 60% (p < 0,001) i 52% (p

> 0,05) po 10 dniach stosowania. Dodatkowo, indukcyjny efekt oceniono dla CYP2D1 o 22% (p=0,008), Mdr1a o 267% (p < 0,0001), Mdr1b o 86% (p < 0,0001), Mrp1 9-krotny (p < 0,0001), Mrp2 o 83% (p < 0,0001) w wątrobie i dla Mrp2 o 35% (p < 0,001) w nabłonku jelita. Znaczny spadek poziomu mRNA w wątrobie obserwowano dla CYP3A1 (ludzki CYP3A4) i Mdr1b w nabłonku jelita. Ponadto, wykazano również nieznaczny spadek w ilości mRNA dla CAR, PXR i ARNT po 3 dniach stosowania ekstraktu.

Wnioski: Wyniki badania sugerują, że Glycine max może zmieniać poziom ekspresji enzymów CYP, szczególnie CYP3A4 i CYP1A1 związanych z biotransformacją ksenobiotyków (leków, prokancerogenów) oraz może uczestni- czyć w klinicznie znaczących interakcjach z lekami metabolizowanymi przez te enzymy. Ponadto, wzrost poziomu mRNA CYP1A1 (ludzki CYP1A1) może nie tylko redukować kancerogenność obcych związków ale może również aktywować niektóre ich kancerogenności. W przypadku transporterów, uważa się, że wzrost ich ekspresji w orga- nizmie może prowadzić do zwiększonej fetoprotekcji, redukcji wrażliwych tkanek (mózg, łożysko) na ekspozycję ksenobiotyków i zmniejszenia efektywności leczenia niektórych chorób. Stąd nadal poszukuje się bezpiecznych substancji, które mogłyby skutecznie modulować aktywność transporterów, szczególnie w leczeniu chorób hormo- no-zależnych np. osteoporozy czy raka piersi, które występują głównie w okresie postmenopauzalnym.

Słowa kluczowe: enzymy CYP / transportery / soja / interakcje / / czynniki transkrypcyjne / poziom ekspresji /

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Materials and Methods Soybean extract

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Ta b l e I . Sequences of primers used for the real-time PCR analysis and PCR conditions.

Gene 3ULPeUVeTXenFe¶ĺ¶ Product

size (bp)

PCR conditions

Cycle Denaturation Annealing Extension CAR F

CAR R

GGA GGA CCA GAT CTC CCT T

GAC CGC ATC TTC CAT CTT GT 130 35 95ºC, 8s 58ºC, 8s 72ºC, 8s

PXR F PXR R

TCC ACT GCA TGC TGA AGA AG

AAC CTG TGT GCA GGA TAGGG 187 35 95ºC, 8s 55ºC, 8s 72ºC, 8s

RXR F RXR R

CCT GAG TTC TCC CAT CAA TG

GAC GCC ATT GAG GCC TAG A 190 35 95ºC, 8s 57ºC, 7s 72ºC, 8s

GR F GR R

CTG GAA TAG GTG CCA AGG CT

CCG TAA TGA CAT CCT GAA GCT 210 40 95ºC, 10s 58ºC, 8s 72ºC, 8s

AHR F AHR R

ATAGCTACTCCACTTCAGCC

TCATGCCACTTTCTCCAGTC 244 35 95ºC, 8s 52ºC, 8s 72ºC, 8s

ARNT F ARNT R

CAG AAC TGT CAG ACA TGG TAC

AGT CAG AGA CAT ACA CCA CTC 246 40 95ºC, 8s 57ºC, 7s 72ºC, 8s

HNF1Į F HNF1Į R

GCT CGG AAG ATG ACA CGG AT

CTT GTT GAG GTG CTG GGA CA 245 35 95ºC, 8s 60ºC, 7s 72ºC, 8s

HNF4Į F HNF4Į R

CTG GAG GAT TAC ATC AAC GAC

GTG TTC TTG CAT CAG GTG AG 164 40 95ºC, 8s 57ºC, 7s 72ºC, 8s

Mdr1b F TGA CGT GAA TGA CGC TGG

207 40 95ºC, 10s 59 ºC, 8s 72ºC, 8s

Mdr1b R CAC ATG GCA GAT GAC AAC C

F – Forward; R – Reverse

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Figure 1. The effect of Glycine max (100 mg/kg) on the expression level of CYP genes in rat liver after 3 and 10 days of treatment. The control group was defined as 100%. Data were presented as mean ± SEM (n = 10). *p<0.05, **p<0.001,

***p<0.0001 as compared to controls (one-way ANOVA test).

Figure 2. The effect of Glycine max (100 mg/kg) on the expression level of transcription factors and ARNT in rat liver after 3 and 10 days of treatment. The control group was defined as 100%. Data were presented as mean ± SEM (n = 10).

*p<0.05, **p<0.001, ***p<0.0001 as compared to controls (one-way ANOVA test).

Figure 3. The effect of Glycine max (100 mg/kg) on the expression level of genes encoding transmembrane transporters in rat liver and intestinal epithelium after 21 days of administration. The control group was defined as 100%. Data were presented as mean ± SEM (n = 10). *p<0.05, **p<0.001, ***p<0.0001 as compared to controls (one-way ANOVA test).

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Oświadczenie autorów

1. Anna Bogacz – wykonanie badań laboratoryjnych, przygotowanie tekstu manuskryptu i piśmiennictwa.

2. Joanna Bartkowiak-Wieczorek – wykonanie badań laboratoryjnych, zebranie literatury, przygotowanie manuskryptu.

3. Przemysław Ł. Mikołajczak – zebranie materiału, współautor tekstu pracy.

4. Beata Rakowska-Mrozikiewicz – opracowanie wyników badań, współautor tekstu pracy.

5. Edmund Grześkowiak – analiza i interpretacja wyników, współautor tekstu pracy.

6. Hubert Wolski – przygotowanie manuskryptu, współautor tekstu pracy.

7. Bogusław Czerny – przygotowanie manuskryptu, współautor tekstu pracy.

8. Przemysław M. Mrozikiewicz – autor koncepcji i założeń pracy, korekta i aktualizacja manuskryptu – autor zgłaszający i odpowiedzialny za manuskrypt.

Źródło finansowania: Praca była finansowana z grantów nr N405 676940 i N405 677140.

Konflikt interesów: Autorzy nie zgłaszają konfliktu interesów oraz nie otrzymali żadnego wynagrodzenia związanego z powstawaniem pracy.

References

1. Potter SM, Baum JA, Teng H, [et al.]. Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr. 1998, 68, 1375-1379.

2. Dennison E, Yoshimura N, Hashimoto T, Cooper C Bone loss in Great Bretain and Japan: a comparative longitudinal study. Bone. 1998, 23, 379-382.

3. Drews K, Seremak-Mrozikiewicz A, Puk E, [et al.]. Efficacy of standardized isoflavones extract (Soyfen) (52-104 mg/24h) in moderate and medium-severe climacteric syndrome. Ginekol Pol.

2007, 78, 307-311.

4. Messina MJ. Legumes and soybeans: overview of their nutritional profiles and health effects.

Am J Clin Nutr 1999, 70, 439-450.

5. Messina MJ, Loprinzi CL. Soy for breast cancer survivors: a critical review of the literature. J Nutr. 2001, 131, 3095-3108.

6. Persky V, Van Horn L. Epidemiology of soy and cancer: perspectives and directions. J Nutr.

1995, 125, 709-712.

7. Anderson JW, Johnstone BM, Cook-Newell ME, Talalay P. Meta-analysis of the effect of soy protein intake on serum lipids. N Engl J Med. 1995, 333, 276-282.

8. Prestera T, Holtzclaw WD, Zhang Y, [et al.]. Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc Natl Acad Sci USA. 1993, 90, 2965-2969.

9. Guengerich FP. Effects of nutritive factors on metabolic processes involving bioactivation and detoxication of chemicals. Ann Rev Nutr. 1984, 4, 207-231.

10. Riddick DS, Lee C, Bhathena A, [et al.]. Transcriptional suppression of cytochrome P450 genes by endogenous and exogenous chemicals. Drug Metab Dispos. 2004, 32, 367-375.

11. Zheng W, Jin F, Dunning LA, [et al.]. Epidemiological study of urinary 6 -hydroxycortisol to cortisol ratios and breast cancer risk. Cancer Epidemiol Biomark Prev. 2001, 10, 237-242.

12. Rushmore TH, Kong AN. Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr Drug Metab. 2002, 3, 481-90.

13. Ribeiro A, Archer A, Beyec JL, [et al.]. Hepatic nuclear factor-4, a key transcription factor at the crossroads between architecture and function of epithelia. Recent patents on endocrine. Metab Immun Drug Discov. 2007, 1, 166-175.

14. Sharom FJ. ABC multidrug transporters: structure, function and role in chemoresistance.

Pharmacogenomics. 2008, 9, 105-127.

15. Morris ME, Zhang S. Flavonoid–drug interactions: Effects of flavonoids on ABC transporters.

Life Sci. 2006, 78, 2116-2130.

16. Sikic BI. Modulation of multidrug resistance: at the threshold. J Clin Oncol. 1993, 11, 1629- 1635.

17. Eichhorn T, Efferth T. P-glycoprotein and its inhibition in tumors by phytochemicals derived from Chinese herbs. J Ethnopharmacol. 2012, 141, 557–570.

18. Mrozikiewicz PM, Bogacz A, Karasiewicz M, [et al.]. The effect of standardized Echinacea purpurea extract on rat cytochrome P450 expression level. Phytomed. 2010, 17, 830-833.

19. Sugamo T, Nakamura K, Tamura H. Effects of cytochrome P450 inducers on the gene expression of ocular xenobiotic metabolizing enzymes in rats. J Health Sci. 2009, 55, 923-929.

20. Andersson U, Grankvist K, Bergenheim AT, [et al.]. Rapid induction of long-lasting drug efflux activity in brain vascular endothelial cells but not malignant glioma following irradiation. Med Oncol. 2002, 19, 1-9.

21. Ronis MJ, Rowlands JC, Hakkak R, Badger TM. Inducibility of hepatic enzymes by 3-methylcholantrene and isosafrole differs in male rats fed diets containing casein, soy protein isolate or whey from concentration to adulthood. J Nutr. 2001, 131, 1180-1188.

22. Helsby NA, Williams J, Kerr D, [et al.]. The isoflavones equol and genistein do not induce xenobiotic-metabolizing enzymes in mouse and in human cells. Xenobiotica. 1997, 27, 587- 596.

23. Helsby NA, Chipman JK, Gescher A, Kerr D. Inhibition of mouse and human CYP 1A- and 2E1 -dependent substrate metabolism by the isoflavonoids genistein and equol. Food Chem Toxicol.

1998, 36, 375-382.

24. Laurenzana EM, Weis CC, Bryant CW, [et al.]. Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism cytochrome P-450 enzymes, and estrogen receptor alpha expression. Food Chem Toxicol. 2002, 40, 53-63.

25. Anderson GD, Rosito G, Mohustsy MA, Elmer GW. Drug interaction potential of soy extract and Panax ginseng. J Clin Pharmacol. 2003, 43, 643-648.

26. Singhal R, Badger TM, Ronis MJ. Rats fed soy protein isolate (SPI) have impaired hepatic CYP1A1 induction by polycyclic aromatic hydrocarbons as a result of interference with aryl hydrocarbon receptor signaling. Toxicol Appl Pharmacol. 2008, 227, 275-283.

27. Li Y, Ross-Viola JS, Shay NF, [et al.]. Human CYP3A4 and murine CYP3A11 are regulated by equol and genistein via the pregnane X receptor in a species-specific manner. J Nutr. 2009, 139, 898-904.