In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9848-9
Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells Yitong Liu & Michael F. Santillo & Thomas J. Flynn & Martine S. Ferguson
Received: 10 September 2014 / Accepted: 9 November 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.
Keywords Genistein . CYP1A . Induction . Inhibition Sex hormones Y. Liu (*) : M. F. Santillo : T. J. Flynn Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Laurel, MD, USA e-mail: [email protected] M. S. Ferguson Division of Public Health Informatics and Analytics, Office of Analytics and Outreach, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD, USA
Introduction Genistein, the most abundant soy isoflavone, has been extensively evaluated in in vitro (Leung et al. 2005), preclinical (Ju et al. 2001; Ju et al. 2002) and clinical studies (Khan et al. 2012) to determine its role in the prevention and treatment of cancer. Although genistein has been reported to reduce cancer incidence in animals and humans (Adlercreutz et al. 1995; Lamartiniere et al. 2002), there are also studies that report the opposite effect (Ju et al. 2001; Ju et al. 2002; Allred et al. 2004). One reason for this discrepancy may be attributable to the biphasic proliferative effect of genistein on estrogen receptor-positive breast cancer cells (Leung et al. 2005). Additionally, cytochrome P450 1A (CYP1A), a liver enzyme that participates in xenobiotic metabolism, may also be responsible for genistein’s conflicting role in cancer prevention and causation. Genistein may modulate CYP1A, an enzyme that can both detoxify (Nebert et al. 2004) and activate (Ma and Lu 2007) carcinogenic chemicals. These conflicting results illustrate the need to understand the effects of genistein on the activity and expression of CYP1A. CYP1A1 is poorly expressed in human liver but is highly inducible, while CYP1A2 is expressed principally in the liver and is also inducible (Moon et al. 2006). The induction of both CYP1As is mediated by aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor (Tompkins and Wallace 2007; Westerink and Schoonen 2007). HepG2 is a cell line derived from a well-differentiated hepatocellular carcinoma of a 15-year-old Caucasian male (Aden et al. 1979; Knowles et al. 1980). HepG2 cells have emerged as a suitable model to study the regulation of CYP1A1 and CYP1A2, at both the transcriptional and enzyme activity levels (Westerink and Schoonen 2007). Confluent HepG2/C3A cells, a clonal derivative of HepG2, are an improved in vitro model for hepatotoxicity studies (Liu et al. 2011). Although genistein has been previously studied as a CYP1A mediator both in vitro (Helsby
LIU ET AL.
et al. 1998; Moon et al. 2006; Jos et al. 2007) and in vivo (Chen et al. 2011), these earlier studies have subtleties in experimental design that complicated the conclusions that were made. For example, incubation times, culture conditions, and the timing of genistein and CYP1A substrate administration in vivo can all lead to mixed effects regarding CYP inhibition and induction. More importantly, no studies have evaluated the potential sex hormone modulation of CYP1A induction and inhibition by genistein despite previous knowledge of genistein’s involvement in female breast cancer and male prostate cancer. Since genistein is widely consumed as a natural phytoestrogen to prevent both breast and prostate cancer (Leung et al. 2005; Ahmad et al. 2013), it is critical to evaluate genistein’s sex hormone-modulated effects on CYP1A activity, which can detoxify or activate carcinogens. The objective of the current study is to investigate the impact of sex hormones and genistein on CYP1A inhibition and induction at both the gene expression and enzyme activity levels. HepG2/C3A cells were cultured in media with defined sex hormone levels and tested for CYP1A inhibition and induction after short-term (immediate) and long-term (72 h) exposures with genistein. A specific CYP1A inducer, β-naphthoflavone (BNF), and inhibitor, α-naphthoflavone (ANF), were also included in the experimental design as positive controls for comparative purposes.
Materials and Methods Materials. HepG2/C3A cells (catalog no. CRL-10741) were obtained from ATCC (Manassas, VA). HepG2/C3A genetic profile was authenticated by confirming alleles for nine different markers including AMEL, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, and vWA in February 2014 (IDEXX BioResearch, Columbia, MO). Cell culture medium and medium supplements were obtained from Life Technologies (Grand Island, NY) and Sigma-Aldrich (St. Louis, MO). Charcoal–dextran stripped fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA). Recombinant human growth hormone was purchased from the National Hormone and Peptide Program (Torrance, CA). The following were obtained from Sigma-Aldrich: testosterone, progesterone, estradiol, 7-ethoxyresorufin, resorufin, Hoechst 33258, salicylamide, dicumarol, genistein (≥98%), ANF (>90%), BNF (>90%), and DMSO. RNeasy Mini Kit and QIA shredder were purchased from QIAGEN (Valencia, CA). Applied Biosystems high-capacity cDNA reverse transcription kit and TaqMan gene expression assay (CYP1A1 assay: Hs00153120_m1); CYP1A2 assay: Hs00167927_m1) were purchased from Life Technologies.
Cell culture. HepG2/C3A cells were cultured in phenol red-free Dulbecco’s minimal essential medium (DMEM) with low glucose, pyruvate, Glutamax, and pyridoxine. This basal medium was supplemented with MEM nonessential amino acids (1%), HEPES (10 mM), and charcoal–dextran stripped fetal bovine serum (10%). Insulin– transferrin–selenium supplement, linoleic acid–oleic acid– albumin supplement, triiodothyronine (10 nM), dexamethasone (20 nM), and recombinant human growth hormone (2 ng/mL) (Dhir et al. 2006) were included in the medium to replace essential growth factors. Antibiotics were not added to the medium. Cell culture medium was supplemented with sex hormones at physiological concentrations to mimic in vivo hormonal environment (Flynn and Ferguson 2010). Male hormone-supplemented culture medium was composed of testosterone (20 nM), progesterone (2 nM), and 17β-estradiol (0.2 nM); female hormonesupplemented culture medium contained testosterone (2 nM), progesterone (2 nM), and 17β-estradiol (0.4 nM). Cells were plated into 96-well tissue culture plates (2.0×104 cells/well) for enzyme activity assay, and into six-well plates (3.0×105 cells/well) for gene expression assay. Plates were incubated at 37°C in 5% CO2. Cells reached confluence in 4–5 d and then treated on culture day 8. Cell treatment. For the long-term induction study, cells were incubated for 72 h with genistein, positive control BNF, or vehicle control, followed by CYP1A activity and gene expression assays. For inhibition studies, cells were first incubated with BNF for 72 h to induce CYP1A activities. This was necessary for accurate inhibition measurements since basal CYP1A expression and activity were relatively low in uninduced HepG2/C3A cells. Cells were then incubated with genistein or the positive control inhibitor ANF, along with CYP1A substrate 7-ethoxyresorufin, for enzyme activity measurement as described below. Assay of total DNA content using Hoechst 33258 reagent after cell lysis (Flynn and Ferguson 2010) was employed as a measure of total cell number. For all treatments, there was less than 15% loss of cell viability as measured by total DNA content. CYP1A activity assay. 7-Ethoxyresorufin O-dealkylase (EROD) assay (Flynn and Ferguson 2010) was used to evaluate CYP1A activity by measuring fluorescence of the product resorufin from the non-fluorescent substrate 7ethoxyresorufin. Further metabolism of resorufin to nonfluorescent products by phase II conjugating enzymes (e.g., sulfo- or glucuronyl-transferases) or by diaphorase was inhibited by inclusion in the assay mixture of salicylamide (1.5 mM) and dicumarol (10 μM), respectively. Fluorescence was measured continuously for 1 h (kinetic assay) using a BMG FLUOstar Omega plate reader.
SEX HORMONE AND GENISTEIN MODULATE CYP1A
Results First, 72-h induction of CYP1A activity by genistein combined with either no-hormones, male, or female hormonesupplemented media was investigated. As genistein increased from 2 to 10 μg/mL (Fig. 1), CYP1A activity significantly increased in all three groups (p
Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells Yitong Liu & Michael F. Santillo & Thomas J. Flynn & Martine S. Ferguson
Received: 10 September 2014 / Accepted: 9 November 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.
Keywords Genistein . CYP1A . Induction . Inhibition Sex hormones Y. Liu (*) : M. F. Santillo : T. J. Flynn Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Laurel, MD, USA e-mail: [email protected] M. S. Ferguson Division of Public Health Informatics and Analytics, Office of Analytics and Outreach, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD, USA
Introduction Genistein, the most abundant soy isoflavone, has been extensively evaluated in in vitro (Leung et al. 2005), preclinical (Ju et al. 2001; Ju et al. 2002) and clinical studies (Khan et al. 2012) to determine its role in the prevention and treatment of cancer. Although genistein has been reported to reduce cancer incidence in animals and humans (Adlercreutz et al. 1995; Lamartiniere et al. 2002), there are also studies that report the opposite effect (Ju et al. 2001; Ju et al. 2002; Allred et al. 2004). One reason for this discrepancy may be attributable to the biphasic proliferative effect of genistein on estrogen receptor-positive breast cancer cells (Leung et al. 2005). Additionally, cytochrome P450 1A (CYP1A), a liver enzyme that participates in xenobiotic metabolism, may also be responsible for genistein’s conflicting role in cancer prevention and causation. Genistein may modulate CYP1A, an enzyme that can both detoxify (Nebert et al. 2004) and activate (Ma and Lu 2007) carcinogenic chemicals. These conflicting results illustrate the need to understand the effects of genistein on the activity and expression of CYP1A. CYP1A1 is poorly expressed in human liver but is highly inducible, while CYP1A2 is expressed principally in the liver and is also inducible (Moon et al. 2006). The induction of both CYP1As is mediated by aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor (Tompkins and Wallace 2007; Westerink and Schoonen 2007). HepG2 is a cell line derived from a well-differentiated hepatocellular carcinoma of a 15-year-old Caucasian male (Aden et al. 1979; Knowles et al. 1980). HepG2 cells have emerged as a suitable model to study the regulation of CYP1A1 and CYP1A2, at both the transcriptional and enzyme activity levels (Westerink and Schoonen 2007). Confluent HepG2/C3A cells, a clonal derivative of HepG2, are an improved in vitro model for hepatotoxicity studies (Liu et al. 2011). Although genistein has been previously studied as a CYP1A mediator both in vitro (Helsby
LIU ET AL.
et al. 1998; Moon et al. 2006; Jos et al. 2007) and in vivo (Chen et al. 2011), these earlier studies have subtleties in experimental design that complicated the conclusions that were made. For example, incubation times, culture conditions, and the timing of genistein and CYP1A substrate administration in vivo can all lead to mixed effects regarding CYP inhibition and induction. More importantly, no studies have evaluated the potential sex hormone modulation of CYP1A induction and inhibition by genistein despite previous knowledge of genistein’s involvement in female breast cancer and male prostate cancer. Since genistein is widely consumed as a natural phytoestrogen to prevent both breast and prostate cancer (Leung et al. 2005; Ahmad et al. 2013), it is critical to evaluate genistein’s sex hormone-modulated effects on CYP1A activity, which can detoxify or activate carcinogens. The objective of the current study is to investigate the impact of sex hormones and genistein on CYP1A inhibition and induction at both the gene expression and enzyme activity levels. HepG2/C3A cells were cultured in media with defined sex hormone levels and tested for CYP1A inhibition and induction after short-term (immediate) and long-term (72 h) exposures with genistein. A specific CYP1A inducer, β-naphthoflavone (BNF), and inhibitor, α-naphthoflavone (ANF), were also included in the experimental design as positive controls for comparative purposes.
Materials and Methods Materials. HepG2/C3A cells (catalog no. CRL-10741) were obtained from ATCC (Manassas, VA). HepG2/C3A genetic profile was authenticated by confirming alleles for nine different markers including AMEL, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, and vWA in February 2014 (IDEXX BioResearch, Columbia, MO). Cell culture medium and medium supplements were obtained from Life Technologies (Grand Island, NY) and Sigma-Aldrich (St. Louis, MO). Charcoal–dextran stripped fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA). Recombinant human growth hormone was purchased from the National Hormone and Peptide Program (Torrance, CA). The following were obtained from Sigma-Aldrich: testosterone, progesterone, estradiol, 7-ethoxyresorufin, resorufin, Hoechst 33258, salicylamide, dicumarol, genistein (≥98%), ANF (>90%), BNF (>90%), and DMSO. RNeasy Mini Kit and QIA shredder were purchased from QIAGEN (Valencia, CA). Applied Biosystems high-capacity cDNA reverse transcription kit and TaqMan gene expression assay (CYP1A1 assay: Hs00153120_m1); CYP1A2 assay: Hs00167927_m1) were purchased from Life Technologies.
Cell culture. HepG2/C3A cells were cultured in phenol red-free Dulbecco’s minimal essential medium (DMEM) with low glucose, pyruvate, Glutamax, and pyridoxine. This basal medium was supplemented with MEM nonessential amino acids (1%), HEPES (10 mM), and charcoal–dextran stripped fetal bovine serum (10%). Insulin– transferrin–selenium supplement, linoleic acid–oleic acid– albumin supplement, triiodothyronine (10 nM), dexamethasone (20 nM), and recombinant human growth hormone (2 ng/mL) (Dhir et al. 2006) were included in the medium to replace essential growth factors. Antibiotics were not added to the medium. Cell culture medium was supplemented with sex hormones at physiological concentrations to mimic in vivo hormonal environment (Flynn and Ferguson 2010). Male hormone-supplemented culture medium was composed of testosterone (20 nM), progesterone (2 nM), and 17β-estradiol (0.2 nM); female hormonesupplemented culture medium contained testosterone (2 nM), progesterone (2 nM), and 17β-estradiol (0.4 nM). Cells were plated into 96-well tissue culture plates (2.0×104 cells/well) for enzyme activity assay, and into six-well plates (3.0×105 cells/well) for gene expression assay. Plates were incubated at 37°C in 5% CO2. Cells reached confluence in 4–5 d and then treated on culture day 8. Cell treatment. For the long-term induction study, cells were incubated for 72 h with genistein, positive control BNF, or vehicle control, followed by CYP1A activity and gene expression assays. For inhibition studies, cells were first incubated with BNF for 72 h to induce CYP1A activities. This was necessary for accurate inhibition measurements since basal CYP1A expression and activity were relatively low in uninduced HepG2/C3A cells. Cells were then incubated with genistein or the positive control inhibitor ANF, along with CYP1A substrate 7-ethoxyresorufin, for enzyme activity measurement as described below. Assay of total DNA content using Hoechst 33258 reagent after cell lysis (Flynn and Ferguson 2010) was employed as a measure of total cell number. For all treatments, there was less than 15% loss of cell viability as measured by total DNA content. CYP1A activity assay. 7-Ethoxyresorufin O-dealkylase (EROD) assay (Flynn and Ferguson 2010) was used to evaluate CYP1A activity by measuring fluorescence of the product resorufin from the non-fluorescent substrate 7ethoxyresorufin. Further metabolism of resorufin to nonfluorescent products by phase II conjugating enzymes (e.g., sulfo- or glucuronyl-transferases) or by diaphorase was inhibited by inclusion in the assay mixture of salicylamide (1.5 mM) and dicumarol (10 μM), respectively. Fluorescence was measured continuously for 1 h (kinetic assay) using a BMG FLUOstar Omega plate reader.
SEX HORMONE AND GENISTEIN MODULATE CYP1A
Results First, 72-h induction of CYP1A activity by genistein combined with either no-hormones, male, or female hormonesupplemented media was investigated. As genistein increased from 2 to 10 μg/mL (Fig. 1), CYP1A activity significantly increased in all three groups (p
