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Isolated ginsenosides aPPD and aPPT induced cytochrome P450 1 A 1 mRNA expression Zhao, Yang 2006

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I S O L A T E D G I N S E N O S I D E S A P P D A N D A P P T I N D U C E D C Y T O C H R O M E P450 1A l M R N A EXPRESSION  by YANG ZHAO B . Med., Shenyang Medical College, 2000  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR THE D E G R E E OF M A S T E R OF SCIENCE  in  T H E F A C U L T Y OF G R A D U A T E STUDIES  (Surgery)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A July 2006  © Yang Zhao, 2006  ABSTRACT Introduction:  Ginseng is commonly used i n herbal preparations for traditional  Chinese medicine.  It contains over twenty ginsenosides amongst which aglycon  protopanaxadiol (aPPD) and aglycon protopanaxatriol (aPPT) have been shown to be the primary circulating metabolites with potent medicinal properties.  To evaluate the  prospect o f metabolic drug interactions the effects o f aPPD and aPPT on expression and function o f human cytochromes P450 C Y P 1 A 1 was assessed. Methods:  Real Time R T - P C R and western blotting were used to measure C Y P 1 A 1  m R N A and protein expression i n human HepG-2 and Caco-2 cells treated with aPPD or aPPT for 12 and 24 hours. C Y P 1 A 1 activity.  Gudluc 1.1 was luciferase labeled and used as the C Y P 1 A 1  promoter constructs. pRL-TK  A P 4 5 0 - G l o ™ C Y P 1 A 1 assay was used to measure  It was co-transfected with A r y l hydrocarbon receptor (AhR) and  (control) plasmids to  pGL3B-CYPlAl  examine  CYP1A1  mRNA  induction v i a A h R .  plasmid was constructed containing longer C Y P 1 A 1  promoter  sequence (-2425, +352) than Gudluc 1.1 (-1301, -819) and used to investigate the induction mechanism outside o f A h R pathway. Results:  There was a dose-dependent induction o f C Y P 1 A 1 m R N A expression in  both HepG-2 and Caco-2 cells dosed for 12h and 24h with either aPPD or aPPT.  The  result was statistically significant at concentrations o f 5 u M and above; however, this was not correlated with increased protein levels.  P 4 5 0 - G l o ™ reporter assay produced  a significant increase i n C Y P 1 A 1 activity only after treatment with 80 and 160 u M ii  aPPD.  N o effects were found at lower concentrations o f aPPD and all concentrations  o f aPPT. activation.  Induction o f C Y P 1 A l m R N A by aPPD and aPPT was independent o f A h R Functional sequence was outside o f the region from -2425 to +352.  Conclusions:  Overall, our results suggest that aPPD and aPPT exert a significant  inductive effect on C Y P 1 A 1 m R N A level, while also activating C Y P 1 A 1 activity only with high concentrations o f aPPD.  protein  This induction o f m R N A level is not  likely to be regulated by A h R .  iii  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F FIGURES  vii  ACKNOWLEDGEMENTS  viii  CHAPTER 1 INTRODUCTION  1  1.1  Ginseng  1  1.1.1  Ginseng History  1  1.1.2  Ginseng Classification and Composition  2  1.1.3  Ginsenoside Structure  3  1.1.4  Ginsenoside Nomenclature  3  1.1.5  Functions and Properties o f Ginseng  4  1.1.6  Functions and Properties o f Ginsenoside  5  1.1.7  Clinical use o f ginseng  6  1.1.8  Metabolism o f Ginsenosides  7  1.2  1.3  Cytochrome P450s  8  1.2.1  Discovery, Nomenclature o f Cytochrome P450s  8  1.2.2  Cytochrome P450 Structure  9  1.2.3  Oxidative Function o f Cytochrome P450s  11  1.2.4  Induction and Inhibition o f C Y P s  12  1.2.5  Cytochrome P450 1A1  14  1.2.6  Ginsenosides and Cytochrome P450s  15  Hypothesis and Objectives  17  iv  C H A P T E R 2 M A T E R I A L S AND M E T H O D S  25  2.1 Cell Culture  25  2.2 Growth Factor and Drug Treatment  25  2.3  2.2.1  Time from C Y P 1 A l m R N A to Protein Determination  26  2.2.2  M T T Procedure  26  2.2.3  Induction Study i n Both Transcriptional and Translational Levels  27  Cell Lysis  27  2.3.1  Total R N A Extraction  27  2.3.2  R N A Concentration Determination  28  2.3.3  Total Protein Extraction  28  2.3.4  Protein Concentration Determination  28  2.4  Genomic DNA Digestion in RNA Sample  29  2.5  Reverse Transcription  30  2.6  Conventional PCR  30  2.7  Real Time PCR  31  2.8  Western Blotting  32  2.9 2.10  2.8.1  Electrophoresis  32  2.8.2  Transfer o f Separated Proteins to Nitrocellulose Membrane  33  2.8.3  Ponceau Red Stain  33  2.8.4  Blocking  34  2.8.5  Primary Antibody Incubation  34  2.8.6  Secondary Antibody Incubation and Detection  34  P450 Glo CYP1 A l Activity Assay  35  Plasmid  37 v  2.11  2.12  2.13  CYP1A1 Promoter Plasmid Cloning  37  2.11.1  Conventional P C R  37  2.11.2  T O P O T A Cloning  38  2.11.3  Restriction Digestion  38  2.11.4  T4 Ligation and S equencing  39  Transfection  40  2.12.1  Optimization Experiment  40  2.12.2  Transfection with Gudluc Plasmid  41  2.12.3  Transfection with p G L 3 B - C y P / ^ / Plasmid  41  Luciferase Assays  42  C H A P T E R 3 RESULTS 3.1  44  Study of CYP1 A l Induction at The Transcriptional and Translational Level..44 3.1.1  Determination o f The Time Required for C Y P 1 A 1 m R N A to Translate  Protein  44  3.1.2 Cytotoxicity Study  45  3.1.3  45  Induction Study i n Transcriptional Level  3.2  Effects of Ginsenoside Treatment on Protein Translation  46  3.3  CYP1 A l Metabolic Activity  47  3.4  Mechanistic Studies of CYP1 A l  47  3.4.1  Gudluc 1.1 plasmid, and Induction o f CYP1A1 by a P P D and aPPT with  AhR  47 3.4.2  p G L 3 B - C T P L 4 7 Plasmid and Induction o f CYP1A1 by a P P D and aPPT ..48  C H A P T E R 4 DISCUSSION  56 vi  REFERENCE  67  LIST O F T A B L E S Table 1.1 Ginseng species  19  LIST O F FIGURES Figure 1.1 The ginseng saponins of protopanaxatriol  20  Figure 1.2 The ginseng saponins of protopanaxadiol  21  Figure 1.3 Thin-layer chromatograms of the saponins of Panax ginseng roots  22  Figure 1.4 The proposed catalytic cycle of cytochrome P450 for hydroxylation reactions 23 Figure 1.5 General CYP1A1 induction model  24  Figure 2.1 Structure of the PGL3B-CYP1 A l plasmid  43  Figure 3.1 Determination of the time required for CYP1A1 mRNA to translate protein 49 Figure 3.2 Cytotoxicity study result for aPPD and aPPT in HepG-2 and Caco-2 cell lines 50 Figure 3.3 Induction study of CYP1A1 by aPPD and aPPT in transcriptional level.51 Figure 3.4 The induction study of CYP1 A l after aPPD and aPPT 24h treatment at translational level 52 Figure 3.5 CYP1A1 metabolic activity study after aPPD and aPPT treatment  53  Figure 3.6 CYP1A1 induction mechanism study related to AhR using Gudluc 1.1 plasmid 54 Figure 3.7 pGL3B-CFPL41 plasmid function test and the use of this construct in CYP1A1 induction mechanism study 55  vn  Acknowledgements  First and foremost, I would like to express m y sincere appreciation to m y two supervisors:  Dr. E m m a Guns and Dr. W i l l i a m Jia.  They provided me not only with  techniques and knowledge i n research, but also with strong moral support.  I feel  very fortunate to have benefited from their immense knowledge and kindness.  I  would also like to thank Dr. Marcel Bally, who offered many useful suggestions throughout m y project.  A special thank you to Dr. Simon Cowell, who was a  post-doctoral fellow i n Dr. Guns' laboratory. endless supply o f ideas.  H e guided me through the initial challenges at the  beginning o f m y research career.  M y deepest thanks to L i n g Tian, who was a  visiting scholar i n Dr. Jia's laboratory.  H e saved me a great deal o f time by teaching  and assisting me with cloning techniques. and Catherine Wood.  H e was very patient and gave me an  M y infinite gratitude to A n d y Eberding  They were always very patient and helpful.  appreciate their help with the early drafts o f this thesis.  I greatly  M y English improved a  great deal from their word-by-word revision.  Finally, warmest thanks to m y family, especially m y mom, for their unconditional love and invaluable spiritual support throughout m y Masters program.  CHAPTER 1 INTRODUCTION 1.1  Ginseng  1.1.1  Ginseng History Ginseng has been used as a popular herbal remedy in A s i a for over 5500 years  (Yun 2001a). it as food.  People i n northern China who struggled with hunger and disease used They discovered that it had healing properties and this knowledge was  passed down through generations.  M a n y myths evolved regarding its medicinal and  healing abilities to restore homeostasis ( H u 1977).  The first written account o f  ginseng was i n a document describing plants and herbs, 'Shennong Bencao Jing' by Tao Hongjing during the Liang Dynasty, 502-557 A D (Yun 2001a). o f this work describes ginseng as follows: "Ginseng Kuei-kai.  is also called Jen-hsien,  It tastes sweetish, and its property is slightly cooling.  gorges of the mountains.  The translation or  It grows in the  It is used for repairing the five viscera, quieting the spirit,  curbing the emotion, stopping agitation, removing noxious influence, brightening the eyes, enlightening the mind, and increasing the wisdom. to longevity with lightweight. "  Continuous use leads one  Shiu-ying H u , a researcher from Harvard University,  translated it i n late 1970s, to make it accessible to people i n the scientific and academic world outside o f China ( H u 1977). Currently, ginseng is still widely used as a health tonic, not only i n A s i a , but  1  throughout the world.  In 1993, it was reported in the U S A that 34% o f the adults in  the U S use at least one alternative therapy (Astin 1998). herbal medicines are the most prevalent.  O f these alternative therapies,  In 1998, total sales i n the U . S . A . for herbal  remedies approached 4 billion U S dollars.  A t the same time, annual sales o f ginseng  were $98 m i l l i o n U S dollars with an annual growth rate o f 26% (Yun 2001a).  Due to  increasing consumption, the role o f ginseng as a medicine has drawn the attention o f numerous researchers.  1.1.2 Ginseng Classification and Composition There are two types o f ginseng commercially available that differ only by method o f preparation:  white ginseng and red ginseng. White ginseng is prepared b y  drying the root after peeling off the skin, while red ginseng is prepared by steaming and drying the root with the skin still intact (Shibata 2001).  The ginseng family contains  many species (Table 1.1), and most herbalists recognize three species as having medicinal properties: Panax japonicus  Panax ginseng  C A . Meyer (Chinese and Korean ginseng),  C A . Meyer (Japanese ginseng), and Panax quinquefolius  (American  ginseng) (Bahrke & Morgan 2000). Several classes o f compounds have been isolated from ginseng root.  These  include triterpene saponins, essential oil-containing polyacetylenes and sesquiterpenes, polysaccharides,  peptidoglycans,  nitrogen-containing  compounds,  and  various  ubiquitous compounds such as fatty acids, carbohydrates, and phenolic compounds (Sticher 1998).  The most active o f these compounds found i n all species o f ginseng  are considered to be the triterpene saponins, which are also called ginsenosides.  To  2  date, thirty-five ginsenosides have been isolated from fresh, white, or red ginseng, o f which 22 are protopanaxadiols, 12 are protopanaxatriols and one, Ro, is an oleanane (Yun 2003).  These ginsenosides vary i n content and relative proportions among  different species o f ginseng (Kitts & H u 2000).  1.1.3 Ginsenoside structure Ginsenosides are believed to be the main pharmacologically active components in ginseng.  Their fundamental skeleton structure is a dammarane-type  triterpene (Figure 1.1 and Figure 1.2). depending  on their  aglycones:  acid-type saponins (Sticher 1998).  tetracyclic  They are categorized i n three groups  protopanaxadiol,  protopanaxatriol  and oleanolic  Nearly all dammarane ginsenosides isolated from  white ginseng root are derivatives o f 20S protopanaxatriol and 20S protopanaxadiol. Ginsenosides isolated from white ginseng are also found i n red ginseng; however, some ginsenosides (20R Rg2, 20R R h i , R h , R s i , Rs2, Q - R i , and N G - R i ) are characteristic 2  saponins found only i n red ginseng(Sticher 1998).  1.1.4 Ginsenoside Nomenclature Ginsenosides are named according to their R f values, and are designated R x , where x=0, a-1, b-1, b-2, b-3, c, d, e, f, 20-gluco-f, g-1, g-2, h-1, etc, starting from lowest R f to highest, as shown i n Figure 1.3.  R f is determined b y thin layer  chromatography R f values o f the different ginsenosides.  3  1.1.5  Functions and Properties of Ginseng Based on the fact that ginsenoside composition is different i n each species, it is  logical to suggest that different species w i l l not have the same pharmacological properties. In fact different species have been suggested to have distinguishing factors throughout historical folklore, for example, i f we consider the ancient A s i a n concept o f the complementary forces o f y i n and yang, it is claimed that North American ginseng provides yin, or a cooling effect to offset stress; while Panax ginseng  C A . Meyer  provides yang, or a warming effect to counter-balance stress (Kitts & H u 2000). Most often, the simple title ginseng is generally considered to mean the Panax ginseng C A . Meyer.  Ginseng has been reported to prevent aging, fatigue, headaches,  amnesia, tuberculosis, diabetes and maladies o f the liver, heart, and kidneys, as well as nervous disorders ( A . 1966; Bittles A H 1979; Popov I M 1973).  It has been used to  treat anaemia, anxiety, shortness o f breath and perspiration, continuous thirst, lack o f sexual desire, dyspepsia, heart pain and nausea (Bahrke & Morgan 1994). Ginseng has been reported to possess non-organ specific preventive effects against various cancers (Yun 2001b).  Chronic consumption is thought to decrease the  incidence o f cancers such as lip, oral cavity and pharynx, larynx, lung, gastric, liver, pancreas, ovarian and colorectal tumors (Shin et al. 2000; Y u n 2001b; Y u n 2003).  An  in vivo study suggested that Panax ginseng may reduce cell damage, especially D N A damage, caused b y gamma- rays ( K i m et al. 1993).  One study reports that American  ginseng extract inhibits breast cancer cell growth in vitro (Duda et al. 1999). Generally, as was described in 'Shennong Bencao Jing', ginseng has been assumed to have low toxicity (Helms 2004) and to possess a variety o f beneficial  4  properties, including anti-inflammatory, antioxidant, and anti-cancer activities, as well as psychological and immune function improvement (Bahrke & Morgan 2000).  1.1.6  Functions and Properties of Ginsenoside A m o n g all the ginsenosides extracted from ginseng, R g , R h , and R h i have 3  been well studied in recent decades.  2  It was reported that Rg3 caused cell cycle arrest  in the G l phase and inhibited cell growth through a caspase-3-mediated apoptosis mechanism ( L i u et al. 2000). lung cancer cells  RJ12 was shown to inhibit in vitro proliferation o f 3 L L  (murine), Morris liver cancer cells (rat), B-16 melanoma cells  (murine), and H e l a cervical cancer cells (human) by causing cells to arrest i n G i phase (Han 1994; Nakata et al. 1998; Shibata 2001).  R h i did not inhibit cancer cell  proliferation but activated adenyl cyclase and promoted melanin synthesis i n melanoma cells, which might be related to reverse transformation o f cancer cells (Shibata 2001). In M a y 2000, a new anti-cancer drug, 'Rg3 Shenyi Jiaonang', appeared on the Chinese market.  Its clinical application was to inhibit tumor angiogenesis and prevent tumor  cell adhesion, invasion, and metastasis.  N o obvious side effects or toxicity have been  reported (Shibata 2001). Previous studies conducted i n our laboratory have demonstrated that 50 mg/kg o f orally administered R h 2 produces significant growth inhibition o f a subcutaneous L N C a P (human prostate cancer metastasis to lymph node) xenograft tumor i n mice (Guns E S 2004).  *Rh2 and paclitaxel were found to act synergistically i n cultured  L N C a P cells to lower both E D 5 0 and E D 7 5 values and produce a significant decrease in both tumor growth and serum P S A (Xie et al. 2006).  5  1.1.7 Clinical use of ginseng Clinical trials have been conducted with ginseng in several different patient populations.  To  assess  the  time-dependent  effects  o f Panax  ginseng  on  health-related quality o f life ( H R Q O L ) , a randomized controlled trial was conducted using a general health status questionnaire (Ellis & Reddy 2002). The improvement i n overall health-related quality o f life cannot conclusively be attributed to ginseng despite  some positive results.  Panax  A double-blind crossover study was  conducted by H o n g et al. i n 2002 to investigate the efficacy o f Korean red ginseng for erectile dysfunction.  In this study, 60% o f the patients answered that Korean red  ginseng improved erectile function (p <0.01) (Hong et al. 2002).  The effect o f eight  popular ginseng types was investigated for postprandial plasma glucose (PG) and insulin (PI) indices were studied by Sievenpiper et al i n 2004. The outcome o f this study suggests that some benefit to taking ginseng may be obtained by diabetics and would be  attributable  to the PPD:PPT-ginsenoside ratio.  Other  unmeasured  ginsenoside or non-ginsenoside components may also be important. (Sievenpiper et al. 2004). I have highlighted only a sample o f some o f the clinical trials which have been conducted with ginseng and numerous others include the evaluation o f its use for fatigue (Elam et al. 2006), neurodegenerative disorders (Radad et al. 2006) and ergogenic properties ( K i m et al. 2005).  C O L D - f X (CVT-E002), a commercial  natural health product manufactured i n Canada by C V Technologies, is used to prevent respiratory infections. It is a proprietary extract o f the roots o f North American ginseng (Panax quinquefolium).  Poly-furanosyl-pyranosyl-saccharides  are the main content and the contents o f polysaccharides and ginsenoside levels are  6  lower and differ significantly from other Asian and American ginseng products (McElhaney et al. 2006).  Ginseng is widely consumed worldwide, highlighting the  importance o f understanding its biological effects, safety and herb-drug interactions. In recent years, several papers reported that cranberry juice had substantial interaction with warfarin (Grant 2004; Rindone & M u r p h y 2006; Suvarna et al. 2003).  Pomelo  juice was also discovered to increase the bioavailability o f cyclosporine, possibly by inhibiting C Y P 3 A or P-gp activity (or both) i n the gut wall (Grenier et al. 2006). These examples emphasize the importance o f also studying the metabolism o f highly consumed ginseng products and their potential interactions with other drugs.  1.1.8 Metabolism of Ginsenosides Ginseng is usually administered orally; therefore, a study o f the metabolism o f ginsenosides i n the digestive system is important.  It was reported that Rbi,Rb2,and  R c were converted into Rg3 under acidic conditions such as those found i n the stomach. R g 3 has been shown to be transformed to 20(S) protopanaxadiol (aPPD) v i a Rh2 by bacteria (including Bacteroides human intestine (Bae et al. 2002).  sp., Eubacterium  sp., and Bifidobacterium  However, Fusobactrium  sp.) in  sp. has only been found to  metabolize Rg3 to RJ12 with no further conversion to aPPD (Bae et al. 2002).  A similar  process converts R g i to protopanaxatriol (aPPT) v i a the ginsenoside R h i (Shibata 2001). These intestinal bacterial metabolites, including compound K , protopanaxadiol, and protopanaxatriol are easily absorbed after deglycosylation i n the stomach and the small intestine and circulate i n the blood as aglycone sapogenins (Hasegawa 2004).  7  Considering that aglycone ginsenosides are the circulating metabolites o f Ginseng products, it is important to investigate their biological activity, as w e l l as their effect on drug metabolism enzymes, such as cytochrome P450s.  1.2  Cytochrome  P450s  A n array o f foreign chemicals (xenobiotics) confront us daily, including environmental contaminants, drugs, carcinogens, etc.  These compounds are often  lipophilic, which facilitates their passage through biological membranes.  They may  accumulate to toxic levels unless they are metabolized to polar, water-soluble products that can be readily excreted from the body.  This biotransformation is often catalyzed  by a group o f enzymes called cytochrome P450s (Denison & Whitlock 1995).  1.2.1 Discovery, nomenclature of Cytochrome P450s Cytochrome P450 enzymes ( C Y P ) were originally discovered b y Klingenberg in rat liver microsomes i n 1958. haem-thiolate proteins (Sato 1964). some prokaryotes.  These enzymes constitute a large super-family o f They are present i n all eukaryotic organisms, and  In eukaryotic organisms, they bind to the endoplasmic reticulum or  mitochondrial inner membranes, whereas in most bacteria they remain dissolved i n the cytosol (Omura 1999).  C Y P enzymes are characterized by an intense  spectral  absorbance peak at 450 nm after being reduced by carbon monoxide and their name derives from this particular characteristic (Sato 1964).  Cytochrome P450 is easily  converted to another solubilized form with an optical absorption peak at 420 nm by anaerobic treatment o f microsomes with snake venom or deoxycholate (Omura 1999;  8  Sato 1964). A standard system o f nomenclature was determined by the P450 Nomenclature Committee ( http://drnelson.utmem.edu/CvtochromeP450.html), ,  amino  acid  sequence  (Danielson 2002).  identity, phylogenetic  association  based on the level o f and  gene  organization  The root for all cytochrome P450 genomic and c D N A sequence  names is an italicized C Y P .  A n Arabic numeral presents for an individual family, and  a letter presents for the subfamily, such as CYP1A  (Danielson 2002).  The same  nomenclature is used for the m R N A and protein sequences except that the designation are not italicized (e.g., C Y P 1 A 1 ) (Danielson 2002).  Members o f the same family  exhibit about 40% amino acid sequence homology, and members o f the same subfamily possess greater than 55% homology (Nelson et al. 1993).  1.2.2  Cytochrome P450 structure Cytochrome P450 enzymes have been found in all living organisms and in most  tissue types.  Their primary function is to modify drugs and other xenobiotics into  more soluble forms, prior to excretion i n urine.  Needless to say, such an abundant and  active enzyme system has garnered a great deal o f interest i n the pharmaceutical industry. Generally, eukaryotic cytochrome P450s range i n size from approximately 480 to 560 amino acids. A l l members o f the cytochrome P450 superfamily share a common globular  to  triangular  structural  framework,  which  consists  o f 2 halves,  the  carboxy-terminal rich i n alpha helices and the amino-terminal rich in beta sheets (Charles A Hasemann 1995).  P450  c a m  (CYP101) o f Pseudomonas putida was the first  9  purified and crystallized P450.  It is a water-soluble bacterial P450, isolated by Dus et  al. in 1970 ( K . Dus 1970) and crystallized in 1974 (Yu & Gunsalus 1974).  To date,  high-resolution crystal structures have been determined for several o f the cytosolic bacterial cytochrome P450s such as C Y P 1 0 1 A 1 , C Y P 1 0 2 A 1 , C Y P 1 0 7 A 1 , C Y P 1 0 8 , C Y P 1 1 1 A 1 , C Y P 1 9 A 1 , C Y P 1 2 1 A 1 , C Y P 1 5 2 , and C Y P 1 7 5 A 1 (Danielson 2002). Eukaryotic P450s have similar structures, except they possess membrane-anchoring regions.  However,  there  are  many  aggregation  products  formed  in  X-ray  crystallography o f eukaryotic C Y P proteins, and it is difficult to obtain single crystals o f them (Guengerich 1993).  In recent decades, research has been performed to  determine crystal structures o f other prokaryotic P450s, and complexes with other compounds.  In 1987, Poulos determined by crystal structure that P 4 5 0  with camphor (Thomas L . Poulos 1987).  carn  complexes  Raag determined X - r a y crystal structures o f  complexes formed b y ferric (Fe ) cytochrome P450 m and different substrates and ca  inhibitors, as well as the ferrous (Fe ) carbon monoxide and camphor bound forms (Raag 1991b; Raag 1989a; Raag 1989b; Raag 1991a). With this information regarding the 3-dimensional structure o f C Y P enzymes, P450BM3 ( C Y P 1 0 2 ) was deemed a good model for membrane bound forms o f the enzymes, and aided the construction o f computer models for numerous microsomal C Y P s including members o f the C Y P 1 A , C Y P 2 A , C Y P 2 B subfamilies and C Y P 3 A 4 (Lewis 1999; Lewis & Lake 1995; Lewis et al. 1999a; Lewis et al. 1999b).  In 2000, the mammalian C Y P 2 C 5 protein was  crystallized b y Williams (Williams et al. 2000).  The crystal structure o f human  C Y P 2 C 9 was subsequently determined i n 2003 (Pamela A . Williams 2003).  In 2004,  Williams et al. identified an unexpected peripheral binding site using three crystal  10  structures o f C Y P 3 A 4 :  unliganded, bound to the inhibitor metyrapone, and bound to  the substrate progesterone.  This active binding site is located above a phenylalanine  cluster, which may be involved i n the initial recognition o f substrates or allosteric effectors (Williams et al. 2004).  1.2.3 Oxidative function of cytochrome P450s Human cytochrome P450 enzymes are found i n most organs throughout the body, but the highest concentrations are found i n the liver (hepatocytes) and small intestine.  The first three C Y P families are the main C Y P families participating in the  metabolism o f xenobiotics.  They are highly expressed within the liver, but are also  expressed in other tissues (Kaminsky 2003; L i n & L u 1998).  Members o f the C Y P  families 2 and 3 are present i n relatively high concentrations i n small intestinal epithelium (Obach et al. 2001; Peters et al. 1991; Zhang Q Y 1999).  C Y P 1 A 1 is  primarily expressed i n extrahepatic tissues, such as lung, small intestine, and placenta (Gonzalez et al. 1992).  C Y P enzymes are essential phase I oxidative enzymes (34)  involved in the metabolism o f fatty acids, steroids, prostaglandins, and environmental pollutants; and the conversion o f procarcinogens and promutagens to deleterious genotoxic compounds (Bernhardt 2006).  In 1996, Coon et al. identified more than 40  different types o f reactions catalyzed by C Y P s , primarily hydroxylations but also including deaminations, desulfurations, dehalogenations, epoxidations, N - , S-, and O-dealkylations, N-oxidations, peroxidations, and sulfoxidations (Coon et al. 1996; Danielson 2002; Omura 1999).  A representative model generally accepted for the  hydroxylation reaction is shown i n Figure 1.4. and may be simplified using the  11  equation:  NAD(P)H  +  H  +  +  O2  +  RH  —>  NAD(P)  +  +  H 0  +  2  ROH  (Danielson 2002)  This reaction involves a series o f electron transfer steps:  ferric P450 reduction,  molecular O2 activation, and the making and breaking o f covalent bonds (Guengerich 1993).  In these monooxygenation reactions, nicotinamide adenine  dinucleotide  phosphate ( N A D P H ) is an electron donor to both microsomal and mitochondrial eukaryotic P450s, whereas nicotinamide adenine electrons  to most  bacterial P450s  (Omura  dinucleotide ( N A D H )  1999).  cytochrome P450 reductase includes two subcomponents:  In endoplasmic  donates  reticulum,  flavin adenine dinucleotide  ( F A D ) and flavin mononucleotide ( F M N ) , which facilitate the direct transfer o f reducing equivalents from N A D P H to the cytochrome P450 heme iron (Danielson 2002).  In the mitochondria, electrons from N A D P H are transferred v i a ferredoxin  reductase to ferredoxin and then to P450 (Nelson 2003).  1.2.4  Induction and inhibition of CYPs Drug-drug interactions are a major concern in pharmacotherapy.  Whenever  two or more drugs are administered over close or overlapping time periods, the possibility o f drug interactions exists. pharmacodynamic  Interactions may be pharmacokinetic and  in nature, pharmacokinetic  interactions  being more  common.  Induction and inhibition o f C Y P enzymes are the most common cause o f documented  12  drug interactions ( L i n & L u 2001).  Inhibition o f C Y P enzymes can be classified  grossly into reversible and irreversible inhibition based on the enzymatic mechanism ( L i n & L u 1998), with reversible inhibition indicated as the most common mechanism in drug-drug interactions (Yan & Caldwell 2001).  A drug with a high affinity for an  enzyme can greatly raise the plasma concentration o f any low affinity metabolized  by the  same  enzyme  and  thereby  drugs  enhance pharmacological and  toxicological effects o f the low affinity drug (Hollenberg 2002).  Enzyme inhibition  occurs quickly and produces almost immediate effects (Hollenberg 2002; L i n & L u 2001).  A number o f factors must be considered with regards to enzyme inhibition.  One o f the most important considerations is the therapeutic index o f the drug.  Patients  who receive drugs with a narrow therapeutic index, anticoagulants, antidepressants or cardiovascular drugs, for instance, are at a much greater risk for drug interactions than patients receiving other types o f drugs ( L i n & L u 1998).  In drug metabolism research,  the term 'induction' means that a substance stimulates the synthesis o f an enzyme whose metabolic capacity is thereby increased.  Induction occurs either due to  increased transcription or translation or as a result o f stabilization o f the enzyme, and is a slow regulatory process compared to inhibition.  It may take days or even weeks for  the full effects to manifest (Hollenberg 2002; L i n & L u 2001).  Enzyme induction may  attenuate the plasma concentration and pharmacological effect o f a drug, which is a substrate o f C Y P enzymes.  Most C Y P enzymes, including human C Y P 1 A l / 2 , 2 A 6 ,  2C9, 2C19, 2 E 1 , and 3 A 4 are inducible (56), but the extent o f induction is variable. Depending on, for instance, which C Y P enzyme is being induced and the compounds that the individual is exposed to, induction may enhance or decrease the toxicity o f the  13  compound.  If the induced C Y P catalyzes the metabolism o f a toxin or carcinogen, the  induction o f the C Y P w i l l increase the capability for metabolic detoxification and elimination, thus is considered a valuable part o f the defense system against exposure to xenobiotics.  Although induction o f the C Y P s may be advantageous in most cases, it  can have a variety o f pharmacological consequences efficacy,  drug-drug  procarcinogens.  interactions,  and  increases  including alterations i n drug  in the  metabolic  activation o f  Consequently, induction o f C Y P s can be viewed as a 'double-edged  sword' for the organism involved (Hollenberg 2002).  1.2.5  Cytochrome P450 1A1 C Y P 1 A 1 has a more significant involvement i n generation o f carcinogenic  metabolites than other C Y P enzymes.  Even though it is expressed at a very low level  in many tissues, such as liver, skin, kidney, lung, it metabolizes a large number o f xenobiotic chemicals to cytotoxic and/or mutagenic derivatives (Denison & Whitlock 1995; Dickins 2004).  These compounds include polycyclic aromatic hydrocarbons  (PAH), which are ubiquitous i n cigarette smoke, city smog and charcoal-cooked foods (Miners J O 2000).  The current literature suggests that, the classical mechanism o f  C Y P 1 A 1 induction is by activation cascade o f the aryl hydrocarbon receptor ( A h R ) ( M a 2001)  A number o f P A H s , including 3-methylcholanthrene,  3,4-benzo(a)pyrene,  and 2,3,7,8-tetrachloro-(p)-dioxin ( T C D D ) were shown to have high affinity for the A h R and also be potent inducers o f C Y P 1 A 1 (Dickins 2004). (a-NF) is a classic C Y P 1 A 1 inhibitor (Taura et al. 2004).  Alpha-naphthoflavone  A h R is normally present in  cytosol as an inactive form associated with two heat shock Hsp90 proteins and another  14  not well characterized protein. After the ligand binding, this complex dissociates and A h R is activated. After its activation, A h R is able to translocate i n the nucleus, where it dimerizes with the A h R nuclear translocator.  This new complex binds specifically to  enhancer D N A sequences within the CYP1A1 promoter called xenobiotic responsive elements ( X R E s ) and stimulates transcription o f the target gene (Delescluse et al. 2000; Dickins 2004; Seree E 2004).  There are exceptions o f the general rule o f C Y P 1 A 1  induction v i a the A h R pathway (Delescluse et al. 2000).  The mechanism needs to be  explored and w i l l be discussed further as part o f this thesis.  1.2.6  Ginsenosides and Cytochrome P450s M a n y studies have been conducted to evaluate the influence o f ginseng on  cytochrome P450 enzymes.  The natural ginsenosides, including R b i R b 2 , Rc,Rd,Re,Rf, ;  or R g i , were not found to inhibit the metabolic activity o f P450 enzymes, such as C Y P 3 A 4 , C Y P 2 D 6 , C Y P 2 C 9 , C Y P 2 A 6 , and C Y P 1 A 2 (Chang et al. 2002; H e & Edeki 2004; Henderson et al. 1999; L i u et al. 2006b).  R h i competitively inhibited the  activity o f C Y P 3 A 4 and slightly stimulated C Y P 2 E l ( L i u et al. 2006a).  A Panax  ginseng extract ( G i l 5 ) and North American ginseng extract ( N A G E ) decreased human recombinant C Y P 1 A 1 , C Y P 1 A 2 , and C Y P I B 1 activities i n a concentration-dependent manner (Henderson et al. 1999).  R b i , R b 2 , Rc.Rd, and Rf inhibited C Y P 1 activities at a  concentration o f 50 jig/ml (Chang et al. 2002).  The intestinal bacterial metabolites,  including Compound K , 20(S)-protopanaxadiol (aPPD), 20(S)-protopanaxatriol (aPPT) all exhibited moderate inhibition against C Y P 2 C 9 activity, and a P P D and aPPT also exhibited potent competitive inhibition against C Y P 3 A 4 activity ( L i u et al. 2006b).  15  This result was confirmed i n our laboratory:  C Y P 3 A 4 , 2C9, 2C19 and 2B6 were  noticeably inhibited by aPPD and aPPT, which have minimal inhibitory effects on C Y P 1 A 2 or C Y P 2 D 6 . Further work is needed to verify the effect o f aPPD and aPPT on C Y P 1 A 1 expression and activity.  This experiment is important for clarifying the relationship  between C Y P 1 A 1 inducibility and susceptibility to chemical carcinogenesis i n ginseng consuming populations.  16  1.3  Hypothesis a n d Objectives  Hypothesis Aglycone ginsenoside aPPD and aPPT can affect hepatocytes  and intestinal cells.  CYP1A1  activity i n human  This regulation can be at the transcriptional,  translational and protein levels.  Objective 1:  To determine the time required for CYP1A1 mRNA to translate  CYP1A1 protein Significance:  It is important to know the time required for C Y P 1 A 1 m R N A to be  translated into protein.  Drug incubation time course w i l l be determined based on this  result for the subsequent experiments.  Objective 2 :  To determine if aPPD and aPPT have a cytotoxic effect on  HepG-2 and Caco-2 cell lines in Significance:  vitro  It is well recognized that animal data is inadequate to predict C Y P  induction i n humans, because both the extent and pattern o f C Y P induction may differ markedly between species.  To this end, there have been significant advances in  human in vitro methods to assess enzyme induction in man (Dickins 2004).  This work  w i l l be conducted in an effort to determine the proper concentrations o f drug for treatment.  17  Objective 3:  To study the impact of aPPD and aPPT on human cytochrome  P450 1A1 Aim 1:  To determine whether aPPD and aPPT induce or decrease CYP1A1 gene  expression in HepG-2 and Caco-2 cell lines Aim 2:  To examine the impact of aPPD and aPPT on translation of CYP1 A l  Aim 3:  To observe effects of aPPD and aPPT on CYP1 A l activity in human liver  microsomes Significance:  It was found that C Y P 1 A 1 has a more significant involvement i n  generation o f carcinogenic metabolites from xenobiotics than other C Y P enzymes. aPPD and aPPT are the major active metabolites o f ginseng formed i n the human gut. It is anticipated that when these three aims are accomplished, the impact o f isolated ginsenosides a P P D and aPPT on C Y P 1 A 1 transcription and translation, as well as protein activity w i l l be determined.  Objective  4:  To explore  the  mechanism of CYP1A1  induction on a  transcriptional level. Significance:  A common model o f C Y P 1 A 1 induction i n literature is based on aryl  hydrocarbon receptor ( A h R ) activation initiating a cascade which results i n the induction o f C Y P 1 A 1 (Ma 2001).  The objective is to determine whether aPPD and  aPPT induce CYP1A1 gene expression v i a the A h R pathway or b y some other means.  18  Table 1.1:  Ginseng species.  Taken from Yun, T. K . 2001  1. Panax ginseng C . A . Meyer (Korean ginseng) 2. Panax japonicus C . A . Meyer (Japanese ginseng) 3. Panax major Ting 4. Panax notoginseng (Burkill) F. H . Chen (Sanchi ginseng) 5. Panax omeiensis J. Wen 6. Panax pseudoginseng  Wallich  7. Panax quinquefolius L . (American ginseng) 8. Panax sinensis J. Wen 9. Panax stipuleanatus H . T. Tsai & K . M . Feng 10. Panax trifolius L . (Dwarf ginseng) 11. Panax wangianus Sun 12. Panx zingiberensis C . Y . W u & K M . Feng 13. Panax vietnamensis H a et Grushv. (Vietnamese ginseng)  19  Saponin/Sapogenin  Ri  R  Re  -Glc-Rha  -Glc  Rf  -Glc-Glc  -H  glc-Rc  -Glc-Glc  -Glc  Rgi  -Glc  -Glc  Rg2  -Glc-Rha  -H  NG-Ri  -Glc-Xyl  -Glc  Rhi  -Glc  -H  aPPT  -H  -H  Figurel.l: The ginseng saponins of protopanaxatriol. O. 1998 (Sticher 1998).  2  Source reference:  Sticher,  20  Saponin/Sapogenin R i  2  Rb,  Glc-Glc  Glc-Glc  Rb  Glc-Glc  Glc-Ara(p)  Rc  Glc-Glc  Glc-Ara(f)  Rd  Glc-Glc  -Glc  mRbi  Glc-Glc-Ma  Glc-Glc  mRb2  Glc-Glc-Ma  Glc-Ara(p)  mRc  Glc-Glc-Ma  Glc-Ara(f)  Rg3  Glc-Glc  -H  Rh  Glc  -H  -H  -H  2  2  aPPD  Figure 1.2:  R  The ginseng saponins of protopanaxadiol.  The variation in the  structure o f saponins is shown i n Figure 1.1 and 1.2. A l l compounds listed are 20S configuration except R g , which is a mixture o f S and R . A comprehensive list o f all saponins is too large to include. Source reference: Sticher, O. 1998 (Sticher 1998) 3  21  •  Ro •o Ra, • O Ra O Rag • o Rb, • o Rb • O Rbg • O 0-R • O Rs, • O Rs? O Ro • o Rd • o Re • 0 NG-Rf 0 R! • O glo-R1O Rg« • • Rh, • •o « o O O o Ro R a R b R e Rf Rfofte R d g  a  2  g  t  o o  O  o  o  o O ° ° o» Q • 2  C H C I j - C H O H - H 0 (14/&('1) s  2  1  2  ;  Ro • o • Raj • o Rag • o Ra • o Rb, • o Rb » o 0 Rbg • 0 Q-R, • o Rs, • o Rs^, • o Rc* • Rd • o Re • a NG-Rf o o Rl • g)c-R1" o Rg, • 0 Rg • Rb, • o e> Ro R a R b i Rc RdReRI R g Rb Rg  a  2  B U O H - A C O C S H S - M J J O (4/1/2. upper layer)  Figure 1.3: Thin-layer chromatograms of the saponins of Panax ginseng roots. The crude saponin fraction was analyzed on a plate o f silica gel 100F254 (Merck) with solvents as indicated. Image taken from Sticher et al. 1998 (Sticher 1998).  22  nativa hexacoordinata • ferric form (low-spin)  Product Release BOH  / d - c ^ r Substrate Binding £^>*\S  BH pentacoordinatG ferric complex (hlgh-apln)  oxyforryl intermodiato ** (tow-spin)  'Cys e* First electron roduction  o-o  pontacoordlnato ferrous complex (Mgh*ptn)  ferric peroxycomptex (low-spin)  'Cys Second electron reduction  i"  hoxacoordinate forrous-02 adduet (low-spin)  WW hexacoordlnato CyS fcrrou»-CO Inhibitor complex (tow-spin)  Figure 1.4: reactions.  The proposed catalytic cycle of cytochrome P450 for hydroxylation Image taken from Danielson, P. B . 2002 (Danielson 2002).  23  Figure 1.5:  General CYP1A1 induction model also known as the AhR signaling  transduction pathway. (Delescluse et al. 2000).  Image was derived  from  Delescluse, C . et al. 2000  24  CHAPTER 2 M A T E R I A L S A N D M E T H O D S 2.1  Cell Culture HepG-2 hepatoma cell line ( A T C C # H B - 8 0 6 5 ™ , Manassas, V A , U S A ) and  Caco-2 human colonic carcinoma cell line ( A T C C # H T B - 3 7 ™ , Manassas, V A , U S A ) were cultured i n Dulbecco's Modification o f Eagle's M e d i u m ( D M E M ; Invitrogen), supplemented with 10% fetal bovine serum ( F B S ; G I B C O ™ , Grand Island, N . Y . ) at 37 °C i n a 5% CO2 environment.  Growth medium was changed every three days.  At  80% confluence b y experience, cells were trypsinized in 0.25% trypsin with E D T A 4 N a (Invitrogen, Burlington, O N , Canada), and 10% o f the cells were subcultured.  2.2  G r o w t h Factor a n d D r u g Treatment After trypsinizing and re-suspending i n growth medium, cells were counted  using a haemocytometer.  In order to maintain the same confluence i n the experiments,  cells were seeded at approximately 30% confluence for both cell lines 24 h prior treatment.  Both aPPD and aPPT were received as gifts from Panagin Pharmaceuticals  Company*, and were dissolved i n 100% ethanol or D M S O . aPPD/aPPT  tested  ranged  from  0  to  The concentrations o f 80  uM  in  a  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( M T T ) assay and from 0 to 40 u M in other experiments.  The concentration range was decided using other  literature based reports o f ginsenoside use i n in vitro assay (Kitts. 2004).  During  * Purity of the compounds was confirmed by Panagin Pharmaceuticals Company using H P L C .  25  treatment with aPPD or aPPT, cells were incubated i n D M E M , supplemented with 2% F B S (Ota T 1991).  2.2.1  To determine the time required for CYP1A1 mRNA to be  translated into CYP1A1 protein 2,3,7,8-tetrachloro-(p)-dioxin ( T C D D ) was obtained Specialties Inc (Brockville,ON, Canada).  from  Chromatographic  HepG-2 cells were treated with 10 n M o f  T C D D for 0, 2, 4, 6, 8, and 12 hours i n duplicate for R N A extraction, while for protein extraction, the treatment duration range was 0, 4, 8, 12, 18, and 24 hours.  This  experiment was repeated two times.  2.2.2  M T T procedure Both cell lines were seeded at 2.5 x 10 cells per well i n 96-well plates. 4  Popovich and Kitts determined the cytotoxicity o f aPPD and aPPT i n Caco-2 cell line in 2004 with reported L C n s o f 50 u M and 300 u M respectively (Kitts. 2004). 5  Therefore concentrations lower than 80 u M were used for both cell lines. Cells were treated i n duplicate with a final concentration at 0, 5, 10, 20, 40, 80 u M o f aPPD and aPPT in D M E M media supplemented with 2% F B S . aPPD/aPPT was 12 and 24 hours.  The duration o f exposure to  After 12 or 24 hours, the treated media was  removed and 100 u L o f 0.5 mg/ml M T T i n serum free media was added, followed by a four-hour period incubation at 37 C i n 5% CO2. To solubilize the formazan crystals after incubation, 100 pi o f lysis buffer (20% S D S , 50% N , N-dimethylformamide, 0.4% glacial acetic acid) were added to each well. The optical density was read at 562 nm 26  absorbance i n a microplate reader and relative survival determined compared to untreated wells. These experiments were repeated three times.  2.2.3  Study of the induction of the CYP1A1 at both a transcriptional  and translational level For both cell lines, 6 x l 0 cells were plated into each well i n 6-well plates. 5  The  duration o f treatment for transcriptional studies was 12 h and 24 h, and 24 h for translational studies.  The concentrations o f aPPD and aPPT used for treatment were 0,  1, 5, 10, 20, and 40 u M .  Transciptional level experiments were repeated three times  no replicates each time, but technical triplicate i n Real Time P C R step (n=3). Translational level experiments were repeated two times with biological triplicate each time.  2.3  Cell lysis  2.3.1  Total R N A extraction TRIzoi®Reagent (Invitrogen, Carlsbad, C A ) was used to lyse cells directly i n a  culture dish by adding 1 m l o f T R I i ® Reagent per well using a 6-well plate. The lysed ZO  samples were incubated at room temperature (RT) for 5 min. After adding 0.2 m l o f chloroform, tubes were capped securely. The tubes were vigorously agitated by hand and then incubated at room temperature for 2 to 3 min. After incubation, samples were centrifuged at 12,000 x g for 15 m i n at 4 C . The aqueous phase was then transferred to a fresh tube. To precipitate R N A , 0.5 m l o f isopropyl alcohol was added into each sample to the aqueous phase, followed by incubation at R T for 10 m i n . The sample was 27  again centrifuged at 12,000 x g for lOmin at 4 °C. The supernatant was removed and the precipitated R N A samples were washed with 1 m l o f 75% ethanol i n H2O b y vortexing for 10 sec at low speed. C.  A final centrifugation was performed at 7,500 g for 5 m i n at 4  The R N A pellet was briefly dried i n the open air at R T for 5 to 10 min, and then  dissolved i n D E P C treated H2O by passing the solution through a pipette tip.  2.3.2  R N A concentration determination Nanodrop-1000  Spectrophotometer  (NanoDrop Technologies,  Wilmington,  Delaware U S A ) was used according to the protocol provided with the instrument.  2.3.3  Total protein extraction Harvested cells were washed with ice-cold Phosphate Buffered Saline (PBS)  and were detached gently from the plate with a rubber scraper on ice into another 0.5 m l o f cold P B S .  The cells were pelleted by centrifuging at 4000 rpm for 4 min.  The  pellets were resuspended i n 100 u L o f R I P A buffer ( N a C l 150 m M , N P - 4 0 1%, N a Deoxycholate 0.5%, S D S 0.1%, Tris (Base) 50mM) supplemented with l x Complete Protease Inhibitor (Roche, Penzberg, Germany).  2.3.4  Protein concentration determination Before quantitation, protein samples were sonicated on ice twice, for 10 sec, at  power level 3 using 550 Sonic Dismembrator (Fisher Scientific). interval o f at least 1 m i n between each sonication. cooled on ice.  There was an  During this interval, samples were  B C A ™ Protein Assay K i t ( P I E R C E , Rockford, IL) was used.  Diluted 28  B S A (bovine serum albumin) standards were prepared at the following concentrations from a 2.0 mg/ml B S A stock standard: 25, 50, 125, 250, 500, 750, 1000, 1500, and 2000 pg/ml.  To prepare the B C A working reagent (WR), 50 parts o f B C A Reagent A  and 1 part o f B C A Reagent B were mixed, followed by a brief vortex to mix.  In order  to be within the range o f the standard curve, all protein samples were diluted 1/10 prior to protein determination. 25 u L o f each standard and unknown sample was pipetted into a flat bottomed 96-well plate i n duplicate.  200 u L o f the W R was then added to each  well, and samples were shaken for 30 s before being covered and incubated at 37 °C for 30 min.  After the incubation, the plate was cooled to RT.  Absorbance was measured  at or near 562 n m on a plate reader (Power Wave, Bio-Tek Instruments, I N C )  2.4  Genomic DNA digestion in RNA sample Deoxyribonuclease I, Amplification Grade was purchased from Invitrogen,  Carlsbad, C A .  Throughout the experiment, DEPC-treated H 0 and RNase-free 2  microcentrifuge tubes were used.  Duplicate tubes were prepared for each sample. One  set was used for the treatment, while the other was used as a negative control (no superscript II enzyme during R T step). at 1 [ig by calculation.  R N A mass was balanced between each sample  The total sample volume was 10 p L .  Initially, R N A sample,  D E P C treated H2O, 10 x reaction buffer and 1 p L o f DNase I were added into the sample and control tubes, followed with an incubation at R T for 15 min.  1 u L o f 25  m M E D T A solution was added into each reaction to inactivate the DNase and then samples were heated for 10 m i n at 65 C .  The R N A samples were ready to use in  reverse transcription.  29  2.5  Reverse Transcription Superscript  Carlsbad, C A .  1M  II R T kit and R N a s e O U T  were purchased from Invitrogen,  Each sample had a no-RT control during R T process.  reaction volume was 20 u L .  The total  After mixing and a 10 sec centrifuge at 5000 rpm for  each component, R N A / p r i m e r mixture was added to sterile 0.5 m l tubes as follows:  1  ug o f total R N A , 1 u L o f d N T P m i x (10 m M each), 1 u L o f Oligo (dT) 12-18(0.5 ug/uL), and using D E P C treated H2O to create a volume o f 12 u.L.  The mixture was  incubated at 65 C for 5 min, and then placed on ice for at least 1 min. mixture was prepared as follows:  A reaction  2 u L o f lOx R T buffer, 2 u L o f 50 m M M g C b , 2 u L  of 0.1 M D T T , and 1 u L o f R N a s e O U T ™ .  7 u L o f this reaction mixture was added  into each R N A / p r i m e r mixture followed by a 10 s vortex and a 30 s centrifugation at 5000 rpm for collection.  The final mixture was incubated at 42 °C for 2 min.  Following incubation, 1 u L o f Superscript II R T was added into each sample tube, while the no R T controls had 1 u L D E P C treated water added to each tube, followed by mixing and incubation at 42 C for 50 min.  The reaction was terminated by heating to  70 °C for 15 m i n and then chilled on ice.  2.6  Conventional PCR The purity o f R T step c D N A samples and negative controls were tested by  conventional P C R . (Carlsbad, C A ) .  Platinum Taq D N A polymerase was purchased from Invitrogen  dNTPs were from S u p e r s c r i p t ™ II R T kit used i n R T step.  The  following primers were used i n the experiment:  30  Beta actin: 5 ' - C G T A C C A C T G G C A T C G T G A T - 3'(Forward) Beta actin: 5'- G T G T T G G C G T A C A G G T C T T T G - 3'(Reverse) The P C R protocol included incubation for 2 m i n at 94 °C; followed by 35 cycles o f 94 °C for 15 s, 58 °C for 30 s, and 68 C for 30 s, with an additional 10 m i n for elongation at 68 °C after the last cycle.  2.7  Real Time PCR S Y B R Green P C R Master M i x was purchased  Warrington, U K .  from  Applied Biosystems,  The primer sets described below were used for the real time P C R  experiments: CYP1A1: 5'- C C T T C G T C C C C T T C A C C A T - 3Xforward) CYP1A1: 5'- G T A A A A G C C T T T C A A A C T T G T G T C T C T -3'(reverse) Beta actin:  5'- G C T C T T T T C C A G C C T T C C T T -3'(forward)  Beta actin:  5'- C G G A T G T C A A C G T C A C A C T T -3' (reverse).  c D N A samples from R T step were diluted 1/5.  2 p L o f the diluted c D N A template  was added into each w e l l o f a M i c r o A m p Optical 96-well Reaction Plate (Applied Biosystems, Foster, C A ) with triplicates o f both CYP1A1 and beta actin gene for each sample.  For the 25-pL-reaction volume, the primer master m i x for each reaction  contained 12.5 u L o f S Y B R Green Master M i x , 1 u L o f forward primer, 1 u L o f reverse primer and 8.5 p L o f d F k O .  23 u L o f the primer master m i x was added in each well.  The plate was sealed with Optical Adhesive Covers (Applied Biosystem, Foster, C A ) . A brief centrifugation was conducted to collect the samples.  Following 2 m i n at 50 °C 31  and 10 m i n at 95 C , the amplification was carried out with 40 cycles o f 15 sec at 95 C and l m i n at 60 °C. This amplification was conducted using a 7900HT Fast Real-Time P C R System (Applied Biosystems, Foster, C A ) . Real time P C R results were calculated using C T values.  Delta C T equals the  value o f the C T value for C Y P 1 A 1 gene minus the C T value for beta actin. delta C T equals the value o f delta C T for a given sample minus the control. delta C T was 2 power minus delta delta C T value.  Delta 2 -delta A  F o l d change was calculated by  determining the ratio o f 2 delta delta C T value for a gievn samples over the control. A  Standard deviation was calculated among replicates.  2.8  Western blotting  2.8.1  Electrophoresis Total protein was standardized to a loading volume o f 30 jag per lane. To ensure  a final volume o f approx 40 u L , the protein sample mixture contained 26 u L o f protein lysate/H 0, 4 u L o f N u P A G E Reducing Agent (10x) (Invitrogen, Carlsbad, C A ) , and 2  10 u L o f N u P A G E L D S Sample Buffer (4*) (Invitrogen, Carlsbad, C A ) .  The samples  were denatured at 100 C for 5 m i n followed by a brief spin at 4 °C. N u P A G E ™ 10% Bis-Tris Gels (Invitrogen, Carlsbad, C A ) were used.  After  the comb was removed from the gel, lanes were rinsed with 1 x NuPAGE® M O P S S D S Running Buffer (50 m M M O P S , 50 m M Tris base, 0.1% S D S , and I m M E D T A , p H 7.7).  The chamber was filled with 800 m l running buffer to submerge the whole  system.  PageRuler ™ Prestained Protein ladder (Fermentas, Burlington, Ontario) was  used as a marker, and 25 u l o f protein samples were loaded i n each well.  The gel was  32  run at 200 V for 50 minutes in Invitrogen X C e l l SureLock  2.8.2  gel apparatus.  Transfer of Separated Proteins to Nitrocellulose Membrane One nitrocellulose membrane ( B I O - R A D , Hercules, C A ) (6 cm x 9 cm), two  fiber pads, and four pieces o f filter paper (7 c m x 9 cm) were prepared to make the transferring sandwich for each gel.  A large container was filled with l x transfer  buffer (with 20% methanol) to a depth o f approx. 5 cm.  One o f the transferring  cassettes was opened and placed in the container with the black panel touching the bottom.  From the bottom to the top, the sequence o f the contents o f the sandwich was  as follows:  a fiber pad, two pieces o f filter paper, gel, a nitrocellulose membrane, two  pieces o f filter paper and a fiber pad.  A l l materials were saturated i n transfer buffer  before being placed into the cassette and all bubbles were removed from between the nitrocellulose membrane and the gel.  After the cassette was closed, it was placed in  one o f the transferring boxes (black side o f the cassette facing the black side o f the box). The transferring box was placed into a transferring container with a stirring bar and an ice pack inside. Transfer buffer was added so that the membranes are covered.  The  transfer was run at 100 v for 1 hour at room temperature or run at 30 v overnight at 4 o  C.  2.8.3  Ponceau Red Stain The membrane was removed from the transferring apparatus, followed by  washing i n P B S twice for 10 min, on a shaker. proteins and ensure a consistent transfer.  Ponceau R e d Stain was used to detect  The membrane was covered b y Ponceau and  33  placed on a shaker for 5 min.  After detection, stain was removed with a destain  solution ( 1 % acetic acid) which was applied for 5min.  Proteins lower than 40 k D a  was cut off and the remaining membrane was washed twice i n P B S on a shaker for 5 min.  2.8.4  Blocking To  minimize  non-specific binding, Odyssey B l o c k i n g  Buffer  Biosciences, Lincoln, Nebraska) was used with 1:1 dilution i n P B S .  (LI-COR  The diluted  blocking buffer was applied to completely cover the membrane and placed on the shaker for 1 hour at room temperature.  2.8.5  Primary Antibody Incubation A 1:1 solution o f 1 x T B S - T (0.2% Tween-20) and Odyssey B l o c k i n g Buffer was  prepared to cover the membrane, and primary antibody C Y P 1 A 1 (B-4) (Santa Cruz, Santa Cruz, California), a mouse monoclonal antibody, was added at a dilution o f 1:300. The membrane was incubated for one hour at room temperature and then overnight at 4 °C.  Vinculin (Sigma, Oakville, Ontario), a monoclonal anti-mouse antibody, was used  as an internal standard primary antibody with a dilution o f 1:5000. incubated at room temperature for one hour.  This was  The membrane was washed i n 1 x T B S - T  (0.2% Tween-20) 4 times for 5 min.  2.8.6  Secondary Antibody Incubation and Detection Fluorescent secondary antibody, A l e x a Fluor® 680 goat anti-mouse I g G (H+L) 34  (Molecular Probes, Eugene, Oregon) was used at 1:5000 dilution with 0.02% S D S , and incubated at room temperature for one hour. (0.2% Tween-20) 4 times for 5 min.  The membrane was washed i n 1 x T B S - T  The membrane was scanned using an O d y s s e y ™  Infrared Imaging System ( L I - C O R Biosciences, Lincoln, Nebraska).  2.9  P450 Glo™ CYP1A1 activity assay P 4 5 0 - G l o ™ C Y P 1 A 1 Assay kit was purchased from Promega, Madison, W I ,  USA.  A n N A D P H regenerating system was used ( B D Gentest™, Oakville, Ontario)  and it included two solutions:  solution A containing 26.1 m M N A D P + , 66 m M  Glucose-6-phosphate, and 66 m M M g C l i n H 0 ( N A D P + and GIC-6-PO4) and solution 2  2  B containing 40 U / m l Glucose-6-phosphate dehydrogenase ( G 6 P D H ) i n 5 m M sodium citrate.  The experiment was repeated twice with technical triplicates each time. Human liver microsomes, containing a mixture o f C Y P proteins, were used  instead o f cell lines.  The P 4 5 0 - G l o ™ C Y P 1 A 1 assay kit consists o f three components,  a specific luminogenic substrate (Luciferin Chloroethyl Ether - Luciferin-CEE), a lyophilized Luciferin Detection Reagent and its reconstitution buffer ( P 4 5 0 - G l o ™ Buffer). human  To assay cytochrome P450 1A1 activity, the substrate was incubated with liver  microsomes  alpha-naphthoflavone  to  generate  the  luciferin  reporter.  TCDD  (ot-NF), a C Y P 1 A 1 protein inducer and a C Y P 1 A 1 activity  inhibitor respectively, were used as controls throughout the experiment. concentrations,  80  and  and  160  u M , were  used,  because  cytotoxicity was  Higher not  a  consideration due to the use o f human liver microsomes. Reconstituted Luciferin Detection Reagent was prepared by equilibrating the  35  P450-Glo  Buffer  and Luciferin  Detection Reagent to room temperature and  transferring the contents o f one bottle o f P450-Glo Buffer (10 ml) to a bottle containing the lyophilized luciferin Detection Reagent.  A homogeneous solution was obtained by  mixing thoroughly. In each reaction, 4 X Cytochrome P450/KPCVSubstrate Reaction Mixture contained 20 p:g o f human liver microsome ( H L M ) , 4 0 0 m M o f KPO4, and 100 u M o f Luciferin-CEE. Corporation  The concentration o f all materials was optimized by Promega  (Technical  Bulletin  No.325).  H L M and  CYP1A1  substrate  Luciferin-CEE were thawed i n a 3 7 C water bath quickly and then kept on ice.  For  each reaction, 12.5 ul o f 4 X Cytochrome P450/KPO4/Substrate Reaction Mixture was added per well i n a 96-well plate.  For negative C Y P control reactions, 4 X  Control/KPOVSubstrate Reaction Mixture was prepared using an equivalent amount o f a preparation that lacked the C Y P 1 A l substrate Luciferin-CEE. Stock  solutions o f aPPD/aPPT/a-NF  in D M S O  were added  at  a  final  concentration o f 0, 1, 5, 10, 20, 40, 80, 160 u M ; while T C D D was added at concentrations o f 0, 1, 5, 10, 20, 40, 80, 160 n M . D M S O volume was balanced between each sample.  10.5 ul o f d H 0 was added to bring the volume to 12.5 ul per  well  plate.  in  a  96-well  2  For  sample  reactions,  12.5  ul  of  the  4x  microsome/KPOVSubstrate Reaction Mixture was added i n each w e l l and mixed gently. For 'minus substrate' control reaction, 12.5 u L o f the 4x microsome/KP04 Mixture was added instead.  100 u L o f d H 0 was added into wells designated as blanks, for signal  background subtraction.  2  o  The plate was pre-incubated at 37 C for 15 min.  2x N A D P H regenerating system was made by mixing 4x solution A and 4x  36  solution B .  The reaction was started b y adding 25 u L o f 2x N A D P H regenerating  system to both the sample and the control wells.  The plate was gently shaken for 30 s  o  and then incubated at 37 C for 30 min.  After the incubation, the same amount o f  substrate was added into the minus substrate control reactions.  To stop the reaction,  50 p L o f reconstituted Luciferin Detection Reagent was added into both the sample and the control reactions, but not added into the blank wells.  After shaking for 30 s, the  plate was incubated at room temperature for 20 m i n to stabilize the luminescent signal. The luminescence was recorded using E G & G B E R T H O L D Microplate Luminometer L B 96 V (Fisher Scientific).  2.10 Plasmid Three plasmids: A h R , p R L - T K and Gudluc 1.1 were obtained as gifts from Dr. Colleen C . Nelson (University o f British Columbia).  The Gudluc 1.1 plasmid  contains the firefly luciferase gene under control o f a portion o f the upstream promoter region (from -1301 to -819) o f the CYP1A1 elements ( X R E M ) (P. M . Garrison 1996). kinase promoter upstream o f Rluc. Renilla  luciferase  from  the  gene containing four dioxin response  The p R L - T K vector contains a thymidine  Rluc is the slightly modified c D N A encoding  sea pansy Renilla  reniformis  (Promega,  Madison,  Wisconsin).  2.11 C Y P 1 A 1 promoter plasmid cloning 2.11.1 Conventional P C R CYP1A1 promoter Plasmid, p G L 3 B - C T P L 4 / (-2425 to +352) was constructed. 37  The following primers were used in the process o f constructing the CYP1A1 promoter sequence: 5'- A C A T G G T A C C C C G A G T G A C C T T G C T T C T C T - 3 ' (forward) 5'- A C G A A G A T C T C T G G G T C C T G A A G T C C T G A A - 3 ' (reverse). Conventional P C R components in 50 u L o f final volume included 0.2 ug o f human genomic D N A , 2 m M M g , 0.1 U / u L o f Platinum Taq D N A polymerase, 0.2 m M o f 2 +  dNTP, and 0.5 m M o f both forward and reverse primer.  The P C R protocol included  incubation for 3 m i n at 94 ° C ; and 10 cycles o f 94 °C for 45 s, 54 °C for 45 s, and 72 °C for 200 s; then, 30 cycles o f 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 200 s followed with an additional 10 m i n at 72 °C for elongation after the last cycle.  2.11.2 T O P O T A Cloning The P C R product was ligated to pCR®2.1-TOPO vector using T O P O T A Cloning® kit (Invitrogen, Carlsbad, C A ) . The ligation product was transformed into competent  cells (Top 10 cells).  After being plated onto a petri dish (100 u.g/ml o f ampicillin i n agar gel), cells were incubated at 3 7 C overnight. 12 clones o f bacteria were randomly chosen and incubated in 2 m l L B medium with 100 |J,g/ml o f ampicillin, shaking at 250 rpm at 37 °C over night.  PureLink™Quick Plasmid Miniprep K i t (Invitrogen, Carlsbad, C A ) was used to  purify the plasmid.  2.11.3 Restriction Digestion After purification, plasmids were tested by running in 1% agarose gel.  Two o f  38  the successful samples were selected for restriction enzyme digestion to double test them. E c o R l (Invitrogen, Carlsbad, C A ) was selected as the restriction enzyme. After confirmation by running the enzyme digestion product i n 1% agarose gel, another restriction enzyme digestion was performed for the T O P O T A cloning product and p G L - 3 basic vector (4.8 kbp) together, using K p n l (Invitrogen, Carlsbad, C A ) and N c o l (Invitrogen, Carlsbad, C A ) sharing N o . 4 buffer from Invitrogen.  A 2.8 kbp  band and a 4.8 kbp band were cut off and purified with QIAquick®Gel Extraction K i t ( Q I A G E N , Mississauga, O N ) .  2.11.4 T4 Ligation and Sequencing Two  purified  products,  CYP1A1  promoter/Kpnl-Ncol  and  pGL-3  b a s i c / K p n l - N c o l , were ligated by T4 ligase (Invitrogen, Carlsbad, C A ) at 25 °C for 2.5 hours with insert/vector molar ratio around 3:1. The ligation product was transformed into Top 10 cells and after amplification plasmids were extracted using QIAfilter™ Plasmid M a x i kit (Qiagen, Mississauga, ON).  Another restriction enzyme digestion was performed with K p n l and N c o l for  the final plasmid. The result was tested on a 1% agarose gel.  The D N A Sequencing Laboratory  (the University o f British Columbia, Canada) conducted the sequencing for T O P O T A cloning product.  39  2.12 Transfection 2.12.1 Optimization experiment To optimize the transfection experiment with respect to the number o f cells, the transfection incubation time, and the ratio o f D N A to Lipofectin® Reagent (Invitrogen, Carlsbad, C A ) experiments were carried out using a 6-well plate.  Each well received  0.3 pg/pL o f the A h R plasmid, 0.3 pg/pL o f the Gudluc 1.1 plasmid, and 0.02 pg/pL o f p R L - T K plasmid.  In an attempt to obtain 40% confluence after 24 h, 4x 10 cells were 5  plated i n each well.  Similar attempts were made to obtain 50%, 60%, 70%, 80%, and  85% confluence after 24h, with 5*10 , 6><10 , 7*10 , 8 x l O and 8.5 x i o cells plated 5  into each well.  5  5  5  5  The volume o f Lipofectin was 6 p L and the incubation time was 6h for  all the wells i n the confluence optimization group.  To optimize the ratio o f  D N A / L i p o f e c t i n , 4, 6, 8, 10, 12, and 16 p L o f Lipofectin were added with a consistent level o f cells at 6 x 1 0 cells per well and an incubation time o f 6 h. 5  F o r the incubation  time optimization group, 6 x 1 0 cells were plated and 6 p L o f Lipofectin was added into s  each well.  The incubation time range was 4, 6, 18, and 24 h.  Lipofectin was diluted with serum free media and kept at room temperature for 30 min.  D N A was also diluted with serum free media and gently mixed with the  Lipofectin dilution. min.  The dilution mixture was kept at room temperature for another 10  It was then diluted again with serum free media to attain a final volume o f 250  p L per well.  The culture media was discarded and the transfection serum free media  solution was added to the cells.  After adequate transfection time, the transfection  media was discarded and the cells were cultured i n D M E M with 10% F B S for another 24h.  Proteins were harvested using Passive Lysis Buffer (from Dual-Luciferase®  40  Reporter Assay System).  After gentle vortexing, the protein samples were stored at  - 8 0 C until the time o f analysis.  2.12.2Transfection with Gudluc Plasmid Based on the result from optimization experiments the following transfection conditions were used i n transfection experiments:  60% confluence by the beginning  o f transfection, 2 (ig/8 u L o f the DNA/reagent ratio, and an incubation time o f 6h. The concentration o f T C D D was determined according to the literature ( X u et al. 2000). HepG-2 cells were plated i n 24-well plates with 1.5 X 10 cells each well. 5  The  following day, cells were transfected with 0.25 u.g o f the A h R plasmid, 0.25 ug o f the Gudluc 1.1 plasmid, and 0.025 \ig o f p R L - T K plasmid i n each well.  After incubating  for 6 hours, cells were treated with 0, 1, 5, 10, 20, 40 u M o f a P P D and aPPT i n D M E M with 2% F B S .  20 n M and 2 n M o f T C D D were the positive controls for the  experimental duplicate.  D M S O was used as a negative control.  incubation lasted for 38 h.  The treatment  The cells were lysed as described i n section 2.11.1.  2.12.3Transfection with pGL3B-CYPlAl The function o f pGL3B-CYPlAl  construct was tested by comparison with the  positive control p G L - 3 promoter (Promega, Madison, WI) and negative control p G L - 3 basic (Promega, Madison, WI) in HepG-2 cells. was as follows:  The amount o f plasmids i n each well  0.3 jj.g p G L - 3 basic, 0.3 jxg p G L - 3 promoter, 0.3 ug  and 0.03 p,g p R L - T K .  pGL3B-CYP!Al,  Transfection procedure was the same as section 2.11.2.  41  The same amount o f pGL3B-CYP!Al  construct was co-transfected with 0.3 pg  A h R plasmid and 0.03 pg p R L - T K i n HepG-2 cells followed by 20 p M a P P D and aPPT treatment for 24h.  The positive control was treated with 2 n M T C D D for 24h, while  negative control was considered as the addition o f the same volume o f D M S O .  2.13 Luciferase assays Luciferase assays were performed using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) and the E G & G Berthold Microplate Luminometer L B 96V (Berthold Technologies, B a d Wildbad).  Luciferase Assay Buffer was added into  Luciferase Assay Substrate and inverted for 5 times by hand. substrate was added to Stop & Glo® buffer.  The protein samples were thawed from  - 8 0 °C and centrifuged at 12,000 rpm for 4min.  20 p L o f the supernatant from each  sample was added i n a 96-well plate for reading. following conditions:  200 u L o f Stop & Glo®  The plate was read using the  Injection P (Luciferase Assay buffer and substrate) delayed 1.6  s and a measurement interval o f 30 s was conducted.  Injection M (Stop & Glo® buffer  and substrate) delayed 10 s and a measurement interval o f 10 s was conducted. both Injection P and M , the volume was 100 p L .  For  The working temperature was 20 C .  42  CYP1A1  F i g u r e 2.1:  Promoter Gene  Structure o f the YGL3B-CYP1A1  plasmid.  This construct contains a  2425 bp fragment from the upstream region o f CYP1A1 gene and the 352 bp down stream sequence including the first exon, and part o f the first intron.  The plasmid  contains a firefly luciferase gene under control o f the CYP1A1 promoter sequence. 43  CHAPTER 3 RESULTS 3.1  Study of CYP1 A l induction at the transcriptional and  translational level 3.1.1 Determination of the time required for CYP1A1 mRNA to translate protein Before investigating the inductive effects o f ginsenosides, it is necessary to determine the rate at which C Y P 1 A l protein is translated. C Y P 1 A l m R N A and protein inducer, was used.  T C D D , a well-known  The treatment duration for R N A  studies was 0, 2, 4, 6, 8, and 12h, while the time for protein studies ranged from 0, 4, 8, 12, 18, to 24h.  The C Y P 1 A l m R N A was induced to the highest level (237±31  fold) at 12h within the chosen time course range (Figure 3.1A).  W i t h respect to  translation, induction was observed from 12h onwards with a 5.32±1.2 fold increase (n=2).  From 12h to 24h, C Y P 1 A 1 protein expression was stable at levels 5 to 6-fold  greater than the controls (Figure 3.1B).  These results were used to determine  treatment times for the following experiments.  12h and 24h were chosen as  appropriate time points.  44  3.1.2  Cytotoxicity study Every cell line has a unique response to a particular drug.  experiments involved were performed using a M T T assay.  The cytotoxicity  HepG-2 and Caco-2  were the two cell lines used and the cytotoxicity o f a P P D and aPPT i n these two cell lines was tested in this step.  Based on observations that C Y P 1 A l protein was stably  expressed 12 h to 24 h after T C D D treatment, and given that the highest level o f C Y P 1 A l m R N A was seen at 12 h, cell viability o f HepG-2 and Caco-2 cells was measured at 12 h and 24 h after treatment with a range o f a P P D and aPPT concentrations.  Cytotoxicity results over a range o f 0 to 80 u M , shown i n F i g u r e  3.2, indicate that aPPT does not have a cytotoxic effect at concentrations o f 40 u M and lower over 12 h and 24 h treatment durations i n either cell line (n=3).  In  contrast, a P P D treatment at 40 u M resulted i n death o f most cells after a 24 h i n both cell lines (n=3).  When treated for 12 h with 40 u M aPPD, half o f the Caco-2 cells  were dead, but almost no effect was seen on HepG-2 cells (n=3).  3.1.3  Induction study in transcriptional level Real time R T - P C R was used to quantitate C Y P 1 A l m R N A expression i n  HepG-2 and Caco-2 cells after a P P D and aPPT treatment.  The Dose dependent  induction o f C Y P 1 A l was observed i n transcriptional levels following both 12h and 24h treatments o f both HepG-2 and Caco-2 cells, with a P P D and aPPT (Figure 3.3). The induction was statistically significant at 5 u M and all concentrations above.  40 45  u M aPPD treatment led to cell death in both Caco-2 and HepG-2 cells at 24h.  At  this concentration and at the 24h time point, aPPD caused 32.25 ± 24.65 fold increase in C Y P 1 A l m R N A level i n HepG-2 cells (n=2).  The reason for the large variation  is only a small amount o f R N A harvested, which was just enough to perform two experimental repeats.  In Caco-2 cells, at 40 u M aPPD, an insufficient amount o f  R N A was available to make c D N A and as a consequence this treatment data was not collected.  However, 20 u M o f aPPD resulted in a clear increase in the expression o f  C Y P 1 A 1 m R N A (n=3).  3.2  Effects of ginsenoside treatment on protein translation In translation level, Western blotting was used to measure the expression o f  C Y P 1 A l protein.  Treatment with a P P D at 40 n M led to cell death in both cell lines,  therefore, insufficient protein was obtained for analysis.  For other dosage levels,  equal masses o f protein were loaded into each lane. W e have determined C Y P 1 A l induction at a transcriptional level with both aPPD and aPPT i n both cell lines by measuring R N A levels.  The induction at the  translational level was present and significant in HepG-2 cells treated with 20 u M aPPD for 24h, which caused a 1.78 ± 0.14 (p=0.02) fold change (n=2).  In Caco-2  cells, the induction o f 1.38 ± 0.2 fold change was not statistically significant (n=2). C Y P 1 A l protein expression was not induced significantly b y aPPT i n HepG-2 cells at any o f the concentrations tested, although expression was decreased b y treatment with 46  20 p M o f aPPT with a 0.69±0.02 (p=0.04) fold change (n=2).  Overall, these results  did not show significant translational induction o f either a P P D or aPPT with any o f the concentrations tested over 24h i n HepG-2 and Caco-2 cell lines (Figure 3.4).  3.3  C Y P 1 A 1 metabolic activity P 4 5 0 - G l o ™ C Y P 1 A 1 Assay kit was used to evaluate the effect o f a P P D and  aPPT on C Y P 1 A l metabolic activity.  The results described i n Figure 3.5 show that  aPPD did not increase the C Y P 1 A l protein activity at concentrations below 40 p M , but only at higher concentrations o f 80 and 160 p M .  aPPT did not increase  C Y P 1 A l activity significantly at any o f the concentrations tested. C Y P 1 A 1 activity at 80 and 160 p M .  T C D D decreased  While T C D D is a good inducer o f C Y P 1 A 1 i n  cells, it was not found to be a good activator.  In comparison, the positive control for  inhibition, a - N F , inhibited C Y P 1 A l activity i n a dose dependent fashion.  3.4  Mechanistic studies of C Y P 1 A l  3.4.1 Gudluc 1.1 plasmid, and induction of CYP1A1 by aPPD and aPPT with AhR Figure 3.6 shows the luciferase assay data for a P P D and aPPT binding to AhR.  The data o f a P P D and aPPT were the average o f the biological duplicate  (n=2), while data for the T C D D positive control was the 2 n M concentration. Luciferase signal o f samples treated with 20 n M T C D D caused 199.81±54.66 (n=l)  fold increase, data not shown here. over Renilla luciferase signal.  A h R / p R L - T K ratio was calculated by firefly  This ratio i n aPPD and aPPT samples was lower than  2.8±0.16 (n=2), which is similar to the negative control D M S O level at 2.1±0.7 (n=2). However, the ratio o f positive control T C D D sample was 22.64±7.3 (n=l).  From  the plot, it was apparent that aPPD and aPPT did not activate the reporter gene expression through A h R .  The results suggest therefore that the induction o f  C Y P 1 A l m R N A by aPPD and aPPT was not regulated through the A h R pathway.  3.4.2 pGL3B-CYP!Al plasmid and induction of CYP1A1 by aPPD and aPPT In pGL3B-CYPlAl  plasmid, CYP1A1 promoter sequence has driven the  reporter gene expression as shown i n F i g u r e 3.7 A . Similar results to those obtained from transfection with Gudluc 1.1 plasmid was obtained i n F i g u r e 3.7 B .  A h R / p R L - T K ratio was 3.11±0.1 for a P P D , 2.99±0.2  for aPPT, 25.16±8.7 for T C D D and 2.97±0.04 for D M S O (n=2).  The results might  suggest that the functional sequence was outside o f the region spanning from -2425 to +352, which is discussed i n C h a p t e r 4.  This also proved that aPPD and aPPT  induced CYP1A1 gene expression through a mechanism unrelated to A h R pathway and verifies that the Gudluc 1.1 plasmid results were indeed positive evidence o f an induction event independent o f the A h R pathway.  48  A.  Figure 3.1: Determination of the time required for CYP1 A l mRNA to translate protein. The level o f C Y P 1 A 1 m R N A was measured b y real time R T - P C R i n HepG-2 cells after 10 n M T C D D treatment with time course ranged from 0 to 12 h. (B) C Y P 1 A l protein was examined by western blotting. Vinculin was used as loading control for cytoplasmic fractions. The ratio o f C Y P 1 A l over Vinculin intensity was quantitated (n=2).  49  A HepG-2 cells 250 -i 200 4  0  5  10  20  40  80  Dosing Concentration (uM)  Figure 3.2: Cytotoxicity study result for aPPD and aPPT in HepG-2 and Caco-2 cell lines. M T T assays were performed i n either (A) HepG-2 cells (n=3) or (B) Caco-2 cells (n=3). Concentration o f aPPD and aPPT ranged from 0 to 80 p M . Treatment duration was 12 h and 24 h. The relative difference in absorbance is used as an indicator o f the number o f cells that are viable i n each group. The results o f the mean absorbance observed in each o f the treatment groups shows that the relative percentage o f surviving cells compared to the untreated control group, which is taken as 100%. 50  A aPPD 60.00 50.00 & 40.00 •S 30.00 I  20.00  PL,  io.or o.o$  5  ~ ~o~  1  "5  20  40  Dosing Concentration (uM)  fif "20" 40~  Dosing Concentration ( y M)  Figure 3.3:  10  - HepG-2 24h  - « — HepG-2 12h  - Caco-2 24h  —X— Caco-2 12h  Induction study of C Y P 1 A 1 by aPPD and aPPT in transcriptional  level. Dosing concentration ranged from 0 to 40 u M . Treatment duration was 12 h and 24 h. The result was quantitated by using C T value o f both C Y P 1 A 1 and beta-actin gene o f each sample to calculate 2 - A A C T value. A l l the samples were compared with control, which was treated with same volume o f 100% ethanol. Student t-test was used and statistically significant is denoted by * (p<0.05) (n=3). The induction was statistically significant at all concentrations above and including 5 uM. A  51  Western blotting image o f HepG-2 a P P D 24h OuM  luM  5uM  lOuM  M L M  20uM  m m~*  «<•:<» mt  **»  mm  CYP1A1  »mi  vinculin  Effect o n C Y P 1 A 1 protein e g r e s s i o n after 24h aPPD/aPPT treatment  - • — Caco-2 aPPD -m— Caco-2 aPPT -*— HepG-2 aPPD -X— HepG-2 aPPT  5  10  Dosing (11M)  Figure 3.4: The induction study of CYP1A1 after aPPD and aPPT 24h treatment at translational level. The result was quantitated by the intensity ratio o f C Y P 1 A l over vinculin, which was a housekeeping protein used as internal control. A l l other sample results were compared to the control, which was treated with same volume o f 100% ethanol. Mouse liver microsome ( M L M ) was used as a positive control i n western blotting. Results were analyzed using student t-test and statistically significant is denoted by * (p<0.05) (n=2).  52  Effect on C Y P 1 A 1 activity after a P P D / a P P T / a - N F / T C D D treatment  Figure 3.5: CYP1A1 metabolic activity study after aPPD and aPPT treatment. a-NF was a positive control for inhibition. The luciferase signal of each sample was compared to negative control, which was treated with same volume o f D M S O . Results were analyzed using Student t-test and statistically significant is denoted by * (p<0.05) (n=2).  53  Gudluc  1.1  plasmid and induction o f  C Y P 1 A 1  b y a P P D and  aPPT  35 30  0  1  5  10  20  40  T C D D DMSO  Dosing Concentration (uM ) and Positive and Negative Control  Figure 3.6:  C Y P 1 A l induction mechanism study related to AhR using Gudluc  1.1 plasmid. The result o f each sample was determined by calculating the ratio o f luciferase signal: firefly luciferase Renilla luciferase Firefly luciferase was produced b y the Gudluc 1.1 plasmid, which contains the upstream promoter region (from -1301 to -819) o f the CYP1A1 gene, while Renilla luciferase was from p R L - T K , the internal control. HepG-2 cells were co-transfected with Gudluc 1.1, A h R and p R L - T K plasmids (n=2).  54  A pGL3B-CYPlAl construct fuction test  o  150.0  ^  i  Jj  100.0  i—'—1  PGL-3 promoter pGL3B-CYPlAl  PGL-3 basic  Plasmid name  B  p G L 3 B - C Y P l A l plasmid and induction o f C Y P l A l b y aPPD and aPPT 40 35  aPPD  aPPT  TCDD  DMSO  Dosing Drug Name  Figure 3.7:  pGL3B-CYPlAl  plasmid function test and the use of this construct  in C Y P 1 A 1 induction mechanism study ( A ) CYP1A1 promoter sequence has driven the firefly reporter gene expression in the pGL3B-CYPlAl responsive element for CYP1A1  plasmid (n=2).  (B) The  induction by a P P D and aPPT was outside o f the  region from -2425 to +352 i n CYP1A1 gene.  HepG-2 cells were co-transfected with  p G L 3 B - C y P 7 ^ 7 , A h R and p R L - T K plasmids (n=2).  55  CHAPTER 4 DISCUSSION  This thesis described a series o f experiments i n which HepG-2 and Caco-2 cells were used as models for the study o f cytochrome P450.  It may be debated as to  whether these cell lines are appropriate models for a cytochrome P450 induction study.  It is w e l l known that results o f in vivo animal experiments are unable to  predict human C Y P induction because interspecies difference i n genomic sequences exist and different C Y P families and subfamilies are also apparent between species (Nelson 2002).  Taking this into consideration, in vitro induction studies using  human hepatoma cell lines or primary human hepatocytes are preferable and likely to be representative o f human pharmacology.  Wilkening et al (2003) compared  primary human hepatocytes and HepG-2 cells recently.  Their work determined that  both provide useful data but are not experimentally equivalent (Wilkening et al. 2003). Human  hepatocytes  were  the  preferred  model  for  studying  metabolic  biotransformation i n human liver because they express a number o f important phase II enzymes which were similar to those i n human liver samples; the expression level o f these enzymes i n HepG-2 differed significantly from that seen i n primary hepatocytes.  However, they found that the up-regulation o f specific genes by probe  substances was similar i n both groups.  To this end, they concluded that HepG-2  cells may be useful to study regulation o f drug-metabolizing enzymes (Wilkening et 56  al. 2003).  Established cell lines have low C Y P expression levels, however, they  have an unlimited life span, a more stable phenotype than primary cultures and are readily available (Rodriguez-Antona et al. 2002).  HepG-2 cells are the most  frequently used and best-characterized human hepatoma cell line.  This cell line is  still regarded as a good in vitro model for C Y P induction studies, since this it retains a variety o f liver-specific metabolic functions (Allen et al. 2001).  Dvorak et al. (2006)  provided the most recent evidence o f this, i n which the HepG-2 cell line was used to study the effect o f colchicine and nocodazole on CYP1A1 gene and protein expression, as well  as protein activity (Dvorak et al.  adenocarcinoma cell line.  2006).  Caco-2 is a colorectal  It exhibits characteristic cytochrome P450 activity as seen  in the colonic epithelium(Lampen et al. 1998; Rosenberg & L e f f 1993).  In 1998,  Lampen et al. compared catalytic activities, protein and mRNA-expression o f cytochrome P450 isoenzymes i n intestinal cell lines.  In their study, several P450  isoenzymes were investigated, including C Y P 1 A 1 , C Y P 1 A 2 , C Y P 2 C 9 / 1 0 , C Y P 2 E 1 and C Y P 3 A .  The results showed that among those tested Caco-2 cells were the only  cell line which expressed C Y P 1 A 1 at both protein and m R N A level, and was able to produce metabolites similar to those observed in in vivo metabolism studies (Lampen et al. 1998).  In summary, we can conclude that HepG-2 and Caco-2 cell lines are  representative models for C Y P induction studies. Northern blotting was used to detect C Y P m R N A by Agarwal et al. i n 1994 (Agarwal et al. 1994).  This method is only semi-quantitative with a non-specific 57  reaction i n hybridization.  Recently, several PCR-based approaches have  been  introduced for the quantitation o f C Y P m R N A , which include reverse transcription P C R (RT-PCR) (Sumida et al. 2000; Sumida et al. 1999), quantitative R T - P C R (Rodriguez-Antona et al. 2000), and real-time P C R (Bowen et al. 2000; Burczynski et  al.  2001).  These  discrimination capability.  techniques  are  extremely  sensitive  with  single-base  However, both R T - P C R and quantitative R T - P C R use  agarose gels for detection o f P C R amplification at the final phase or end-point o f the P C R reaction, which leads to inaccurate quantification o f m R N A .  In real time P C R ,  exact doubling o f product is accumulating at every cycle, which is known as the exponential phase and provides a distinct advantage over traditional P C R detection. Thus, real time P C R is more quantitative than conventional P C R .  Western blotting  is still a basic tool for investigating gene regulation at the protein level, however, it is not effective for detecting rapid protein degradation.  In this situation, western  blotting is not the best technique chosen. In 2000, Lekas et al. determined that C Y P 1 A l m R N A was rapidly degraded in HepG-2 cells, with a half-life o f 2.4 ± 0.13 h (Lekas et al. 2000).  They also  generated a model suggesting that loss o f the poly (A) tail is an early step in the degradation o f the C Y P 1 A 1 m R N A (Lekas et al. 2000).  To determine the time  required for C Y P 1 A l m R N A translation to protein, cells should be lysed after a time course, which starts at the time o f removal o f inducer.  However, considering the  rapid degradation o f C Y P 1 A 1 m R N A , T C D D , the inducer, was not removed from 58  cells i n a time course experiment following progression from m R N A to protein for the purpose o f this thesis research.  A s a consequence, the rate o f C Y P 1 A 1 m R N A  translation was not clearly determined, however, the results  obtained i n the  time-course experiments provided a gross time course for drug treatment i n the induction study that followed. The induction study described in this thesis illustrates that the induction function o f C Y P 1 A 1 was only observed at the transcriptional level, but was not i n translational protein expression.  The expression o f many genes are known to be  regulated after transcription, so it is reasonable to say that an increase i n m R N A concentration does not imply an increase in protein expression. is provided (Hashimoto et al. 2006).  A n example o f this  Expression profiles o f melanogenesis-related  genes and proteins i n acquired melanocytic nevus were studied. Pmel-17/gpl00 protein was seen only i n the basal layer.  In that study,  The protein and m R N A  profiles were observed to be appreciably different (Hashimoto et al. 2006).  Many  factors interfere with the process o f R N A degradation and translation to protein.  To  explain the inconsistency o f C Y P 1 A l m R N A and protein expression i n this study, the following rationale might be considered: A possibility would be that a P P D and aPPT increases C Y P 1 A 1 protein degradation after induction by magnifying the production of its protease. Induction  of  CYP1A1  is  commonly  measured  through  increased  Ethoxyresorufin (O) dealkylation ( E R O D ) activity (Chang et al. 2001; Merchant et al. 59  1992;  Shimada et al. 2002; Zhi-Hua Chen 2005)  In this reaction, the substrate  ethoxyresorufin is hydrolyzed to resorufin, a stable and fluorescent compound. Enzyme kinetics are recorded by a fluorimeter.  P450 G l o ™ C Y P 1 A 1 assay was  used i n this study, which provided a luminescent method for measuring C Y P 1 A 1 activity.  The reaction is performed b y incubating human liver microsomes with  luminogenic C Y P 1 A 1 substrate (Luciferin 6' chloroethyl ether (Luciferin-CEE)). The amount o f luminescent light produced is directly proportional to the activity o f CYP1A1.  The advantage o f the P 4 5 0 - G l o ™ C Y P 1 A 1 assay is there are no  fluorescent  excitation and emission overlaps between  analytes, N A D P H  and  cytochrome P450 substrates.  Such an overlap may confound analysis and present  misleading or irrelevant data.  This method is simple, highly sensitive, and specific,  while providing a stable signal with a half-life o f greater than 2 hours. Chang et al. (2002) conducted a previous study on the impact o f ginseng on CYP I family activity. ginseng  They found that ginseng extracts, such as G i l 5 {Panax  extract) and N A G E {Panax Quinquefolius  extract) were able to decrease  human recombinant C Y P 1 A 1 activities i n a dose dependent manner and these inhibitory effects were not identified to be due to any o f the seven ginsenosides: R b l , Rb2, R c , R d , Re, Rf, or R g l , which together account for >90% o f the total ginsenoside content i n ginseng extract.  These inhibitory effects could have been the  result o f non-ginsenoside compound (Chang et al. 2002). 7-ethoxyresorufin O-dealkylation assay was used.  In their experiments, a  To date, the results stated i n this 60  thesis is the first report about the impact o f isolated gisenosides aPPD and aPPT on C Y P 1 A l activity, which was increased at high concentrations o f aPPD at 80 and 160 u M , but not lower concentrations including 40 u M .  Even though these high  concentrations may not be pharmacologically attainable unless the compound is accumulated i n the tissue, this result is still interesting and holds a value as mechanism to study C Y P 1 A l activation.  T C D D , a potent C Y P 1 A 1 inducer, did not  activate C Y P 1 A 1 activity i n human liver microsomes.  It induced C Y P 1 A 1 m R N A  and protein expression indirectly by the A h R (Anderson et al. 2006; Riddick et al. 1994; Shimada et al. 2003), but it is not an activator o f C Y P 1 A l directly.  However,  aPPD could have acted as an activator o f C Y P 1 A 1 , increasing C Y P 1 A 1 activity directly.  To discuss the mechanism, some basic pharmacology definition should be  noted here:  Affinity and intrinsic activity are independent properties o f drugs.  Agonists have both affinity, that is, the ability to bind to the receptor, and intrinsic activity, the  ability to produce a measurable  effect.  dissociation constant and is the reciprocal o f the affinity. greater the affinity.  K d is the equilibrium The smaller the K d , the  The ability o f a drug to produce a physiological effect is  dependent on both receptor  occupancy (which is in turn governed by drug  concentration and K d value) and the propensity o f the drug to activate the receptor. Based on the above theory, i f aPPD binds to a receptor, which triggers C Y P 1 A 1 activity directly, aPPD may have a high K d value, and subsequently a lower affinity, so that higher concentrations o f aPPD are needed to activate C Y P 1 A 1 activity. 61  Another possibility is that aPPD may be a substrate o f C Y P 1 A 1 or its metabolite a P P D - X may be an allosteric activator o f C Y P 1 A 1 .  If this is the case, a P P D might  compete with Luciferin-CEE as the substrate o f C Y P 1 A 1 i n the P 4 5 0 - G l o ™ C Y P 1 A l assay. by luciferin.  Such a competition would result i n a lower luciferase signal caused  This reduction would be complemented by the effect o f a P P D - X ,  which acts as an activator o f C Y P 1 A 1 , subsequently increasing the metabolism o f Luciferin-CEE.  A s the a P P D concentration increases, more and more a P P D - X is  produced; producing an increased C Y P 1 A l activity and resulting in an increase o f the metabolism o f Luciferin-CEE resulting in the production o f more free luciferin. This might be the reason for lack o f induction at low concentrations o f a P P D whereas at higher concentrations the enzyme was induced. The A h R pathway is a well recognized mechanism and regulator o f C Y P 1 A 1 induction at the transcriptional level(Dvorak et al. 2006; Jr. 1999; M a 2001; Seree E 2004).  A s part o f this thesis, the question o f whether aPPD and aPPT are ligands o f  A h R was answered.  Gudluc 1.1 plasmid which contains a portion o f the upstream  promoter region (from -1301 to -819) o f the mouse CYP1A1  gene contains four  dioxin response elements ( X R E M ) (El-Fouly 1994; Fisher 1989; Fisher 1990; P. M . Garrison 1996).  Garrison et al. constructed this plasmid i n 1996 (P. M . Garrison  1996) and it has since been used i n several studies (Abnet et al. 1999; Jeon & Esser 2000; V r z a l et al. 2005).  The results o f this study demonstrate that both aPPD and  aPPT do not activate A h R as indicated by the fact that they did not increase the firefly 62  luciferase signal.  This suggests that the induction o f C Y P 1 A 1 b y a P P D and aPPT is  not regulated b y the A h R pathway. It is possible that aPPD and aPPT may induce C Y P 1 A 1 by activating other receptors, or binding to the CYP1A1 promoter directly. If this is the case, it is possible that the CYP1A1 promoter sequence i n G u d l u c l . l plasmid is too short, resulting i n ineffective binding to the promotor.  Several  different CYP1A1 promoter plasmids have been used to study the A h R pathway and are reported in the literature (Anderson et al. 2006; Shimada et al. 2002; Zhi-Hua Chen 2005).  In 2001, Lee and Safe studied the inhibition o f C Y P 1 A l expression by  resveratrol i n breast cancer cells (Shimada et al. 2002).  In their study, the plasmid  used had the -1142 to +2434 regulatory region from the human CYP1A1 gene fused to the bacterial CAT reporter gene (Shimada et al. 2002).  In 1993, Postlind  constructed p L l A 1 N plasmid, which contained a fragment from -1612 to +292 (all o f exon one, a portion o f intron one and 1612 bp o f 5'-flanking sequences) o f the human CYP1A1 gene. Chen 2005).  Contained within this fragment are three consensus X R E s (Zhi-Hua In 2004, Galijatovic et al. developed a transgenic mouse line, which  carries this p L l A 1 N plasmid, to study the mechanism associated with A h R control o f the CYP1A1  gene in vivo (Anderson et al. 2006).  Based on the p L I A I N plasmid  structure, a longer human CYP1A1 promoter construct, p G L 3 B - l A l (-2425 to +352), was created.  A h R , p G L 3 B - / ^ / , and p R L - T K plasmids were co-transfected into  HepG-2 cells and the resulting luciferase signal was detected.  The result obtained  was the same as that observed using Gudluc 1.1 plasmid, which confirmed the 63  conclusion that the induction o f C Y P 1 A 1 by aPPD and aPPT was not through A h R pathway.  This result also suggested that i f aPPD and aPPT induced the  CYP1A1  gene by binding directly to the promoter, the control sequence might be outside the region from -2425 to +352.  It is also testimony to the effectiveness o f the Gudluc  1.1 plasmid as a good construct for A h R pathway study.  However, there are further  issues to consider when using the p G L 3 B - L 4 7 plasmid.  It is possible that there is a  suppressor region within this part o f the sequence; as a result, the promoter would not be triggered.  This study does not have data to support this hypothesis, but i n future  studies it would be beneficial to truncate the CYP1A1  promoter construct to  investigate the responsive element and the possible existing suppressor. Is C Y P 1 A 1  induction always related to the A h R signaling pathway?  Delescluse (2000) asks this question i n the paper's title (Delescluse et al. 2000). Several exceptions o f C Y P 1 A 1 induction v i a the A h R pathway were reported. Stress conditions such as hyperoxia was found to induce C Y P 1 A 1 (Hazinski et al. 1995; Okamoto et al. 1993). induction does not always require a ligand. compounds have shown they activate CYP1A1,  expression  This demonstrates that C Y P 1 A 1  Several non-polycyclic and non-planar even though they do not compete for  the T C D D binding site on A h R ( A i x et al. 1994; Daujat et al. 1992; Fontaine et al. 1999; Gradelet et al. 1997; Lee et al. 1996; Lesca et al. 1995).  Seree, et al. provided  evidence for another new human CYP1A1 regulation pathway involving peroxisome proliferator-activated receptor-a (PPAR-a) and two peroxisome proliferator response 64  element ( P P R E ) sites which are located within the CYP1A1 promoter (positions -931/-919 and -531/-519) (Seree et al. 2004).  Their results indicated that P P A R - a  ligands, which are common environmental compounds, induced human C Y P 1 A 1 m R N A expression.  Norihito et al. determined a region from - 6 1 4 to - 4 5 8 ( X R E M )  in the CYP1A1 promoter, which is important for C Y P 1 A 1 induction. found  several activators o f human pregnane X receptor  They also  ( h P X R ) and  human  constitutive androstane receptor ( h C A R ) could dramatically induce CYP1A1 promoter activity through 1A1 X R E M .  Their results support a novel role for h P X R and  h C A R i n the regulation o f human CYP1A1 gene (Norihito S 2005). Post-transcriptional stabilization is another possible mechanism o f C Y P 1 A 1 induction by a P P D and aPPT.  The half-life o f C Y P 1 A 1 m R N A may have been  prolonged and subsequently increased the gene expression level as measured due to improvement o f m R N A stability, not the increase o f transcription.  There is still  much to be investigated regarding C Y P 1 A l induction mechanism. In conclusion, the results described in this thesis indicate that a P P D and aPPT induce C Y P 1 A 1 m R N A expression significantly, but that the effect did not carry through to protein translation.  A t the highest concentrations, 80 and 160 u M , aPPD  and aPPT increased C Y P 1 A 1 activity.  Finally, we can also conclude that regulation  of C Y P 1 A l m R N A expression did not occur v i a the A h R pathway. The effect o f aPPD and aPPT on cytochrome P450 3 A 4 expression was initially measured at the transcriptional level as well as C Y P 1 A 1 .  In the H e p G - 2 65  cell line, 12h treatment only slightly induced the expression o f C Y P 3 A 4 with 1,5, 10 u M o f both aPPD and aPPT and 2 0 u M aPPT, but significantly suppressed C Y P 3 A 4 expression with 20, 4 0 u M aPPD and 4 0 u M aPPT.  In the Caco-2 cell line, only 1,5,  10 u M a P P D and l u M aPPT slightly induced C Y P 3 A 4 after 12h treatment.  No  induction o f C Y P 3 A 4 was seen i n other concentrations with both aPPT and aPPD. This result provided further evidences that aPPD and aPPT specifically induced C Y P 1 A 1 m R N A expression. The result i n this thesis indicated that commercially available ginseng products  are  likely to be safe  drugs/environmental  chemicals.  and not  cause problematic interactions  The amount  with  o f circulating ginseng derived  compounds are not likely to be as high as 80 and 160 p M , concentrations o f aPPD and aPPT which have been shown here to activate C Y P 1 A l protein activity in vitro. Further study is necessary i n this area and an important, yet unexplored area would be more thorough identification o f the mechanism o f C Y P 1 A 1 induction.  mRNA  Another aspect that may also play a substantial role in the increase in  C Y P 1 A l m R N A levels is a possible decrease i n the degradation rate o f the transcripts, plausibly through  post-transcriptional  stabilization.  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