JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 JPETThis Fast article Forward. has not been Published copyedited and on formatted. April 3, The 2003 final asversion DOI:10.1124/jpet.102.048645 may differ from this version.
JPET #48645
In Vivo Effect of PC-SPES on Prostate Growth and Hepatic CYP3A Expression in Rats
Teri Wadsworth, Hataya Poonyagariyagorn, Elinore Sullivan, Dennis Koop and Charles E. Roselli.
Downloaded from
Department of Physiology and Pharmacology
Oregon Health & Science University, Portland, OR. jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645
Running Title: In vivo effects of PC-SPES
Correspondence: Dr. Charles E. Roselli, Department of Physiology and Pharmacology L334,
Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098,
Tel (503) 494-5837, FAX (503) 494-4352, email: [email protected]
Number of text pages: 20 number of tables: 4 Downloaded from number of figures: 4 number of references: 40 jpet.aspetjournals.org number of words in the Abstract: 250 number of words the Introduction: 748
number of words in the Discussion: 1478 at ASPET Journals on September 26, 2021 nonstandard abbreviations: National Institute of Diabetes and Digestive and Kidney Disease
(NIDDK); U.S. Food and Drug Administration (FDA); testosterone (T); dihyrotestosterone
(DHT); estradiol (E); 6β-hydroxytestosterone (T-6β-OH); 7α-hydroxytestosterone (T-7α-OH);
16α-hydroxytestosterone (T-16α-OH); 16β-hydroxytestosterone (T-16β-OH); diethylstilbestrol
(DES); cytochrome P450 (CYP); high pressure liquid chromatography (HPLC); liquid chromatography-mass spectrometry (LC-MS); gas chromatography/mass spectrometry (GC/MS);
10 mM Tris-HCl, pH 7.4, 200 mM sodium chloride and 0.1% Tween-20 (TBS-T); radioimmunoassay (RIA); 3,5-dicarboethyoxy-2,6-dimethyl-4-ethyl-1,4-dhydropyridine (DDEP) recommended section assignment: Toxicology
2 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 Abstract
PC-SPES, a proprietary mixture composed of eight different herbs, is used worldwide as
an alternative treatment by prostate cancer patients. It has been suggested that the clinical and in vitro anti-tumor activity exhibited by PC-SPES may be due to estrogenic activity, which in turn may be mediated by the presence of undeclared prescription drug contaminants. Here, we evaluated the in vivo effects of two different commercial lots of PC-SPES in male and female rats. Downloaded from Our high pressure liquid chromatography analysis coupled with gas chromatography/mass spectrometry analysis by an independent lab suggested that PC-SPES lot #5430125 was contaminated with diethylstilbestrol (DES), while lot #5431249 was not. Treatment of male rats jpet.aspetjournals.org with PC-SPES lot #5430125 or DES alone reduced the weight of androgen target organs and decreased circulating levels of sex steroids and lutenizing hormone, while lot #5431249 was at ASPET Journals on September 26, 2021 without effect. In addition, lot #5430125 and DES, but not lot #5431249 increased uterine weight in female rats. These results suggest that the inhibitory effects on androgen targets are mediated through suppression of the hypothalamic-pituitary axis and this suppression is probably due to
DES contamination. We assessed the effects of both lots of PC-SPES and DES on hepatic cytochrome P450 (CYP) expression and activity. Both lots of PC-SPES and DES reduced
CYP3A activity and protein levels. Since the response of CYP3A to PC-SPES was not dependent on whether it contained DES, a phytochemical component of PC-SPES is most likely responsible for this effect. Inhibition of CYP3A has important implications for potential herbal-drug interactions.
3 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 In recent years, the use of complementary or alternative medicine by cancer patients has increased dramatically, with herbal medicines being the most common form of alternative treatment (Eisenberg et al., 1998). Rather than replacing traditional therapy, most herbal treatment
is used in conjunction with it. Herbal preparations are sold over the counter as dietary supplements, prospective regulation by the Food and Drug Administration (FDA) is not required
(Talalay and Talalay, 2001). Therefore, very little scientific information is available regarding Downloaded from effectiveness, mechanism of action, side effects and the potential for interactions with other medications.
Many components of herbal remedies are potential substrates for cytochrome P450 (CYP), jpet.aspetjournals.org a family of enzymes which catalyze the phase I detoxification of both endo- and xenobiotics.
Several studies illustrate that constituents from plants modulate CYP enzyme activity. An at ASPET Journals on September 26, 2021 example is the inhibition of intestinal CYP3A by grapefruit juice, resulting in increased oral availability of drugs such as cyclosporine and felodipine (Bailey et al., 1998). In contrast, St.
John=s wort, an herbal remedy commonly used for depression, contains a potent inducer of
CYP3A. Repeated administration of St. John's wort decreases cyclosporin concentrations in transplant patients and reduces plasma concentrations of digoxin and HIV-1 protease inhibitors in healthy volunteers (Piscitelli et al.; 2000; Mai et al., 2000; Johne et al., 1999). In a screening of 21 commercial ethanolic extracts of commonly used herbal remedies, approximately 75% of the products inhibited CYP3A metabolite formation in vitro (Budzinski et al., 2000). According to a
recent review, 4 of the 7 top-selling herbal medicines in this country interfere with the metabolism
or efficacy of approved synthetic drugs (Izzo and Ernst, 2001). Taken together, these findings demonstrate the potential for drug interactions with herbs that are administered concomitantly with
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JPET #48645 other medications and illustrate the need for studies evaluating the effects of herbal medicines on drug metabolizing enzymes.
PC-SPES is a proprietary herbal mixture used as an alternative form of treatment by prostate cancer patients. It is composed of the extracts of eight different herbs: Dendrantherma morifolium, Tzvel; Ganoderma lucidium, Karst; Glycyrrhiza glabra L: Isatis indigotica, Fort;
Panax pseudo-ginseng, Wall; Rabdosia rubescens; Scutellaria baicalensis, Georgi and Serenoa Downloaded from repens. PC-SPES is considered a form of combination therapy, in which individual herbal components are thought to act synergistically to exert anti-tumor actions (Darzynkiewicz et al.,
2000). Individual components of PC-SPES include flavonoids, glycans, phytoestrogens, jpet.aspetjournals.org polysaccharides, sterols, fatty acids and saponins. Several of these compounds exhibit anti- proliferative, antitumor, antimutagenic, analgesic and/or differentiation-inducing activity (Chen, at ASPET Journals on September 26, 2021 2001). PC-SPES has been shown to mediate antiproliferative effects on prostate cancer cells in vivo and in vitro (Darzynkiewicz, et al., 2000), and reduce PSA levels in some individuals with prostate cancer (Oh et al., 2001; Small et al., 2000). While the mechanism of action of PC-SPES has not been fully elucidated, it has been suggested that the clinical and in vitro effects may be attributed to its estrogenic activity. Side effects include nipple tenderness, gynecomastia and decreased libido (DiPaola et al., 1998). Molecular activities include in vitro downregulation of the antiapoptotic protein bcl-2, estrogen receptor-α, and androgen receptor (Hsieh and Wu, 2002;
Hsieh et al., 1997; Kubota et al., 2000). Although phytoestrogenic components may be responsible for some of these effects, it is also likely that effects may be mediated by the presence of undeclared prescription drug contaminants. In 2002, the FDA warned consumers to stop taking
PC-SPES because it contains undeclared prescription drug ingredients, including the synthetic
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JPET #48645 estrogen diethylstilbesterol (DES). The manufacturer voluntarily recalled PC SPES nationwide
(California Department of Health, 2002).
In these studies, we evaluated the in vivo effects of two different commercial lots of PC-
SPES in male and female rats. Gas chromatography/mass spectroscopy (GC/MS) analysis of lot
#5430125 by an independent lab indicates that this lot contains 25 µg/capsule of DES (California
Department of Health, 2002). Lot #5431249 does not contain this impurity. We evaluated the Downloaded from
effects of oral administration of both lots of PC-SPES as well as DES on the weights of the
prostate and other androgen target organs in male rats. We measured serum concentrations of jpet.aspetjournals.org steroid and gonadotropin hormones to determine whether PC-SPES acts by suppressing the
hypothalamic-anterior pituitary-testicular axis and compared the effect of treatment with PC-SPES
and DES on uterine weights to evaluate whether the effects of PC-SPES could be attributed at ASPET Journals on September 26, 2021 entirely to its estrogenic activity. We also assessed the effects of PC-SPES and DES on hepatic
CYP expression and activity in order to identify potential drug interactions in patients with
prostate cancer that use PC-SPES concomitantly with other medications.
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JPET #48645 Materials and Methods
11-α-hydroxyprogesterone was purchased from Aldrich Chemical Company, Inc (Saint
Louis, MO). PC-SPES (lot 5430125 and lot 5431249) was obtained from BotanicLab (Brea, CA).
Bio-Rad Protein Assay and Molecular Analyst software were obtained from Bio-Rad Laboratories
(Hercules, CA). γ-32P-ATP and GeneScreenJ hybridizaion transfer membranes were obtained from DuPont-NEN (Boston, MA). TRIzol7 Reagent, oligonucleotide probes and T4 kinase were Downloaded from obtained from Invitrogen Life technologies (Carlsbad, CA). Supersignal7 Ultra chemiluminescent substrate was obtained from Pierce (Rockford, IL). G-25 Quick Spin Columns were purchased jpet.aspetjournals.org from Roche Diagnostics Corporation (Indianapolis, IN). Nitrocellulose membrane was obtained from Schleicher & Schuell (Keene, NH). Diethylstilbestrol (DES), Denhardt=s reagent, EDTA, and NADP (grade III) were obtained from Sigma (St Louis, MO). Testosterone (T), at ASPET Journals on September 26, 2021 dihyrotestosterone (DHT), estradiol (E), 6β-hydroxytestosterone (T-6β-OH), 7α- hydroxytestosterone (T-7α-OH), 16α-hydroxytestosterone (T-16α-OH) and 16β- hydroxytestosterone (T-16β-OH) were purchased from Stearaloids, Inc. (Wilton, NH). Rabbit anti rat CYP2B1 and CYP3A2 were generously provided by Dr. James Halpert (University of Texas
Medical Branch, Galveston, TX). Goat anti rat CYP4A1 was a generous gift from Dr. Gordon
Gibson (University of Surrey Guildford, Surrey England). Rabbit anti rat CYP2C11 was provided by Dr. Edward Morgan (Emory University, Atlanta, GA) and rabbit anti mouse 2A5 was provided by Dr. Xin Xin Ding (Wadsworth Center, Albany, NY).
Animals. 40 adult Male Sprague-Dawley rats (200-220 g) and 40 juvenile female (120-
140 g) Sprague-Dawley rats purchased from Simonsen Laboratories (Gilroy, CA) were used in the
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JPET #48645 study. They were maintained on 12-h light and 12-dark cycles and had free access to food and water at all times. Rats were allowed to acclimatize in the facilities for at least 7 days before use.
Treatments. Treatments were given orally by gavage in 1.5% carboxymethylcellulose with 0.2% Tween-20 and were performed daily between 0800-1000 for 10 days. Rats were weighed and examined daily for general health (morbidity and mortality) and checked for signs of fluid aspiration. All procedures were approved by the Institutional Animal Care and Use Downloaded from Committee of Oregon Health & Science University.
Effect of PC-SPES on ventral prostate growth and hormone secretion. Male rats were randomized to one of the following treatment groups: 1) vehicle (n=13), 2) PC-SPES lot jpet.aspetjournals.org
#5430125: 50 mg/kg/day (n=7), 3) PC-SPES lot #5430125: 250 mg/kg/day (n=7), 4) PC-SPES lot
#5431249: 250 mg/kg/day (n=6), or 5) DES: 37.5 µg/kg/day (n= 7). Rats were exsanguinated by at ASPET Journals on September 26, 2021 decapitation the morning after the last treatment and trunk blood collected for steroid hormone and
gonadotropin determinations. The ventral prostate, liver, seminal vesicles, levator ani, anterior
pituitary, adrenal, kidney and spleen were collected, weighed and immediately frozen and stored at
-80oC.
Effect of PC-SPES on uterine weight (estrogen bioassay). Female rats were
ovariectomized and allowed to recover for 2 weeks. At the beginning of the third week, female
rats were placed on a phytoestrogen-free diet (PMI, St. Louis, MO) and randomized to one of the
following treatment groups: 1) control (n=13), 2) PC-SPES lot #5430125: 250 mg/kg/day (n=8),
3) PC-SPES lot #5431249: 250 mg/kg/day (n=6), or 4) DES: 37.5 µg/kg/day (n=13). The morning
after the last treatment, rats were exsanguinated by decapitation and the uterus was dissected and
weighed after all fluid was expressed. The degree of uterine growth is proportional to the dose of
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JPET #48645 estrogen exposure ( Zarrow et al., 1964).
Hormone Measurements. T, DHT and LH were quantified in serum by radioimmunoassay (RIA) after extraction with ethyl ether and chromatography on Sephadex LH-
20 columns as described previously (Hess and Resko, 1973). Assay extraction efficiency for was
66% for T and 75% for DHT. Intraassay coefficients of variation were 3.4% for T and 9.9% for
DHT. The levels of sensitivity were 2 pg/tube for T and 1.8 pg/tube for DHT. Concentrations of Downloaded from LH were determined by RIA using materials supplied by the National Institute of Diabetes and
Digestive and Kidney Disease (NIDDK). Reagents were provided by Dr. A.F. Parlow, Scientific
Director of the National Hormone and Peptide Program of the NIDDK. The standard used in this jpet.aspetjournals.org assay was LH RP-3 and the level of sensitivity was 10 pg/tube. The intraassay coefficient of variation was 4.7%. at ASPET Journals on September 26, 2021 High Pressure Liquid Chromatography (HPLC) Analysis of PC-SPES. HPLC was used to characterize the composition of the two lots of PC-SPES used in these experiments. Fresh ethanolic extracts were prepared by suspending 300 mg of PC-SPES in 1 ml of 70% ethanol. The suspension was shaken at room temperature for 1 h, centrifuged at 1000 x g for 10 min and the supernatant filtered through 0.20 µm nylon membranes. Aliquots (10 µl) of the clarified extracts
were injected on a Waters HPLC system (Millford, MA) consisting of a model 717 plus
autosampler, dual 510 solvent pumps, and a model 486 absorbance detector. Components were
resolved at room temperature on a 150 mm x 4.6 mm C-18 column (Supelco, Bellefonte, PA)
preceded by a 10 mm x 4.3 mm C-18 guard column (Upchurch Scientific, Oak Harbor, WA). A
linear gradient from 90% solvent A (H2O with 0.25% glacial acetic acid) to 100% solvent B
(acetonitrile with 0.25% acetic acid) was delivered over 60 minutes at 0.5 ml/min. Absorbance of
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JPET #48645 the eluate was monitored at 254 nm. In addition, 10 µl aliquots of pure samples of DES (40
µg/ml), warfarin (60 µg/ml) and PC-SPES spiked with DES or warfarin were also analyzed with
this system. For all HPLC analysis, the Millennium32 software system (Waters, Milford, MA) was used for instrument control, data acquisition and processing.
The stability of the PC-SPES suspensions utilized for gavage treatments was assessed by extracting 1 ml aliquots of the suspension with 2 volumes of ethyl acetate at the beginning and end Downloaded from of treatment. The organic layer was removed and dried under reduced pressure. The dried extract was dissolved in 100 µl of 70% ethanol, filtered through 0.2 µm nylon filters and analyzed by jpet.aspetjournals.org HPLC as described above.
Microsome isolation. Microsomes were isolated from rat livers by differential
centrifugation as previously described (Koop et al., 1997). Briefly, liver tissue (250-300 mg) was at ASPET Journals on September 26, 2021 homogenized in 3.5 volumes of ice cold homogenization buffer (10 mM Tris hydrochloride buffer,
pH 7.4, containing 0.25 M sucrose and 1mM EDTA). Samples were then centrifuged in a
Beckman TL-100 ultracentrifuge at 10,000 x g for 20 min at 4oC. The supernatant was collected
and centrifuged at 100,000 x g for 1 h at 4oC. The supernatant was discarded and the pellet
resuspended in 1 volume of microsome storage buffer (10 mM Tris hydrochloride buffer, pH 7.5
containing 20% glycerol). Protein concentrations were determined using the Bio-Rad Protein
Assay and microsomes were stored at -80oC until assayed.
CYP Activity. T hydroxylation was assayed by HPLC as previously described (Brunner et al., 1998). In brief, reaction mixtures contained 0.1 M potassium phosphate, pH 7.4, 0.3 mg microsomal protein, 250 µM T (dissolved in methanol, final methanol concentration did not
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JPET #48645 exceed 0.7% v/v) and an NADPH regeneration system consisting of 0.5 mM NADP, 10 mM
glucose-6-phosphate and 10 mM magnesium chloride. The total reaction volume was 1 ml.
Mixtures were pre-incubated at 37oC for 3 min and the reactions initiated by the addition of 5 U of
glucose-6-phosphate dehydrogenase. Incubations proceeded for 15 min and were quenched by the
addition of 5 ml of dichloromethane. The internal standard (3.6 nmol of 11α-
hydroxyprogesterone) was added, the samples vortexed for 30 sec, then centrifuged at 2000 x g for Downloaded from 5 min at 4oC. The organic layer was removed and dried under reduced pressure. Dried extracts were dissolved in 200 µl T solvent A (methanol/water/acetonitrile, 39:60:1) and analyzed by
HPLC. Extracts (50 µl) were injected on the Waters HPLC system Described above. Metabolites jpet.aspetjournals.org were resolved at room temperature. A concave gradient (curve #6) from 90% T solvent A to 85%
T solvent B (methanol/water/acetonitrile, 80:18:2) was delivered over 22 minutes at a flow rate of at ASPET Journals on September 26, 2021 0.5 ml/min. An 8-min washout of 90% T solvent A preceded each analysis. Absorbance was
monitored at 238 nm. T metabolites were quantitated by comparison of peak area ratios
(metabolite/internal standard) with those generated with authentic standards. Rates were
determined under conditions that were linear with protein and time (data not shown). The
Millennium32 software system (Waters, Milford, MA) was used for instrument control, data aquisition and processing. Chlorzoxazone hydroxylation was analyzed as previously described by
Barmada et al. (Barmada et al., 1995).
Northern Blot Analysis. Total RNA was isolated from rat liver with TRIzol7 reagent as specified by the manufacturer. RNA was quantified spectrophotometrically (A260) and 30 µg was
subjected to northern blot analysis. Briefly, total RNA was electrophoresed in 1% agarose/15%
formaldehyde gels, transferred overnight to GeneScreenJ membranes, cross linked to the
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JPET #48645 membrane by UV irradiation and baked at 80oC for 1 h. Membranes were prehybridized for 1 h at
55oC in hybridization buffer (5X SSC, 1% SDS, 1X Denhardt=s reagent and 0.5M sodium phosphate buffer, pH 6.5) then hybridized overnight in the same buffer containing 1 x 106 cpm/ml
of γ-32P-ATP-labeled oligonucleotide probe (table IV). Following hybridization, filters were washed four times, 15 min per wash, in 5X SSC, 0.1% SDS at 55oC. The presence of comparable
amounts of total RNA in each lane was verified by hybridization of membranes with an Downloaded from
oligonucleotide probe for 18S rRNA. Radioactivity was detected with a GS-363 Bio-Rad
Molecular Imager with a BI imaging screen and signal intensity quantified with Bio-Rad jpet.aspetjournals.org Molecular Analyst software.
Western Blot Analysis. Western blot analysis of microsomal fractions was used to screen for the presence of distinct CYP isozymes. Proteins (20 µg per lane) were separated by SDS- at ASPET Journals on September 26, 2021 PAGE using 9% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were blocked 1
h in blocking buffer (5% non-fat dry milk dissolved in 10 mM Tris-HCl, pH 7.4, 200 mM sodium
chloride and 0.1% Tween-20 (TBS-T)). Blocked nitrocellulose blots were incubated 1 h with
primary antibody in 1% bovine serum albumin (BSA) and TBS-T, washed in TBS-T, then
incubated 30 min with a 1:10,000 dilution of the appropriate secondary antibody coupled with
horseradish peroxidase in blocking buffer. After washing, the immune complexes were detected
by enhanced chemiluminescence (ECL) using Supersignal7 Ultra chemiluminescent substrate.
The following dilutions of the primary antibodies were used: CYP2A1, 1:1000; CYP3A,
1:10,000; CYP 4A1, 1:2500; CYP2B1, 1:5000; CYP2C11, 1:600; CYP2E1, 1:5,000.
Statistics. Data were analyzed using GB-STAT statistical software, version 7.0 (Dynamic
Microsystem, Inc., Silver Spring, MD). Tissue weights and hormone concentrations were
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JPET #48645 analyzed by either t-tests (comparisons between two groups) or One-way ANOVA followed by
Fisher's least square difference posthoc test (comparisons between multiple groups). All western blot and northern blot statistics were expressed as percent of control, and analyzed by t-tests (two- tailed). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
13 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 Results
HPLC Analysis of PC-SPES. HPLC was used to characterize the composition of 70% ethanolic extracts of PC-SPES that was administered in these studies (Fig 1). Lot #5430125 and lot #5431249 exhibited significantly different HPLC profiles. While the overall pattern of UV detectable peaks was similar, very striking differences were noted when comparing area ratios for different peaks. While our analysis did not identify the compound(s) eluting with each peak, the Downloaded from differences we observed suggest that PC-SPES constituents in each lot are present in very different ratios. In addition, lot #5430125 exhibited a unique peak at a retention time of 39 min, which co-eluted with DES (DES standard profile not shown). In addition, when the ethanolic jpet.aspetjournals.org extract from this lot was spiked with 40 µg/ml of DES, the peak was increased with no evident shoulder. DES contamination in this lot was confirmed by gas chromatography-mass at ASPET Journals on September 26, 2021 spectroscopy (GC/MS) analysis by the California Health Department and the level was estimated to be 25 µg/capsule (California Department of Health, 2002). Another peak at 38 minutes, present in both lots of PC-SPES, was found to co-elute with a pure sample of warfarin (warfarin standard profile not shown). The presence of warfarin in both lots was also confirmed by the California
Health Department using liquid chromatography-mass spectrometry (LC-MS) and the estimated level was 80-200 µg/capsule. HPLC profiles of suspension extracts of individual lots were identical on day 1 and day 10 of treatment (data not shown), suggesting the PC-SPES suspensions utilized for gavage were stable over the course of the treatment.
Effect of PC-SPES Treatment on Body and Organ Weights. As shown in Table 1, animals treated with 250 mg/kg PC-SPES lot #5430125 or DES exhibited a significant decrease in
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JPET #48645 the ventral prostate and seminal vesicle relative weights when compared to control treatment.
DES and 250 mg/kg PC-SPES lot #5430125 treatment significantly increased relative kidney
weights. In contrast, PC-SPES lot #5431249 had no effect on the relative weights of the ventral
prostate or other androgen target organs, yet decreased body weight and relative spleen weight.
The weights of the levator ani, anterior pituitatary, adrenal gland were not affected by DES or
either lot of PC-SPES. Downloaded from Effect of PC-SPES on Gonadal Steroids and Gonadotropin Hormone Levels. As shown in table 2, PC-SPES lot #5430125 at a dose of 250 mg/kg significantly decreased circulating concentrations of LH and showed a trend towards a decrease in T and DHT jpet.aspetjournals.org concentrations when compared with control. PC-SPES lot #5431249 had no effect on these hormones. After DES treatment, there was a significant decrease in concentrations of T, a trend at ASPET Journals on September 26, 2021 towards decreased LH concentrations, but surprisingly, an increase in concentrations of DHT.
Effect of PC-SPES Treatment on Uterine Weight. As shown in Table 3, PC-SPES lot
#5430125 but not lot #5431249 exerted significant estrogenic action in the rat uterine weight bioassay. Rats treated with PC-SPES lot #5430125 or DES exhibited a significant increase in uterine weight when compared to control treatment while PC-SPES lot #5431249 had no effect.
Both lots of PC-SPES and DES decreased body weight when compared to control.
Effect of PC-SPES and DES Treatment on Hepatic CYP Activity. The effect two different doses of PC-SPES lot #5430125, PC-SPES lot #5431249 and DES treatment on microsomal testosterone metabolism is shown in Fig 2A. Both lots of PC-SPES at a dose of 250 mg/kg and DES at a dose of 37.5 µg/kg significantly decreased the production of 6β- hydroxytestosterone (T-6β-OH), a functional marker of CYP3A activity. As shown in Fig 2B,
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JPET #48645 DES significantly inhibited the production of 16α-hydroxytestosterone (T-16α-OH), an activity
catalyzed predominantly by CYP2C11. Neither lot of PC-SPES affected CYP2C11 catalyzed T-
16α-hydroxylase activity. No significant treatment effects were observed on the formation of 16β-
hydroxytestosterone (T-16β-OH), or 7α-hydroxytestosterone (T-7α-OH) indicative of no change in
the activities of CYP2B or CYP2A1 (data not shown). PC-SPES treatment did not significantly
affect chlorzoxazone hydroxylation, a functional marker of CYP2E1 activity (data not shown). Downloaded from
Effect of PC-SPES Treatment on CYP Protein. Western blot analysis of hepatic
microsomes was used to monitor the effect of two doses of PC-SPES lot #5430125 (Fig 3A), PC- jpet.aspetjournals.org SPES lot #5431249 (Fig 3B) and DES (Fig 3C) treatment of male rats on CYP3A and CYP2C11
protein levels. Quantification of western blots is consistent with results from T hydroxylation. A
significant decrease in CYP3A protein was observed in microsomes from rats treated with the at ASPET Journals on September 26, 2021 higher dose of PC-SPES lot #5430125, PC-SPES lot #5431249 and DES that is consistent with
the decrease in T-6β-hydroxylase activity. As predicted from the T-16α-hydroxylase results, PC-
SPES did not affect CYP2C11 protein levels. Although not statistically significant, there was a
trend towards decreased CYP2C11 protein levels after DES treatment. This loss was more
evident in the significant suppression of T-16α-hydroxylase activity. None of the treatments affected levels of CYP2E1 or CYP4A1 protein (data not shown).
Effects of PC-SPES & DES Treatment on Expression of CYP3A mRNA. Northern blot analysis utilizing oligonucleotide probes (Table 4) was used to evaluate CYP3A2 (Fig 4A) and CYP3A18 (Fig 4B) mRNA expression in the livers of male rats treated with vehicle, PC-
SPES lot #5430125, PC-SPES lot #5431249, or DES. The intensities of the 2.1 kb CYP3A
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JPET #48645 mRNA transcripts were normalized to 18S rRNA. While there was a trend towards inhibition of
CYP3A2 expression with all treatments, the decrease was not statistically significant. Neither lot of PC-SPES affected CYP3A18 mRNA levels when compared to control. CYP3A9 mRNA was not detected in any of the samples (data not shown). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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JPET #48645 Discussion
In the present study, we evaluated the in vivo effects of two different commercial lots of
PC-SPES. Our results demonstrate that PC-SPES lot #5430125 exhibited significant estrogenic
activity, but lot #5431249 did not. HPLC analysis identified a peak that co-eluted with DES, a
potent synthetic estrogen that was present in lot #5430125 only. In vivo treatment with PC-SPES lot #5430125 or DES, but not lot #5431249, stimulated uterine wet weight in ovariectomized Downloaded from female rats and suppressed prostate growth in male rats. The effect exerted on prostate growth appeared to be mediated through inhibition of the hypothalamic-pituitary-gonadal axis because
treatment with PC-SPES lot #5430125 or DES also suppressed serum concentrations of T and LH. jpet.aspetjournals.org
Conversely, both lots of PC-SPES used in this study as well as DES significantly decreased
CYP3A protein levels and activity, but only DES suppressed CYPC11 activity. Since the at ASPET Journals on September 26, 2021 response of CYP3A to PC-SPES was not dependent on whether it contained DES, we conclude
that a phytochemical component of PC-SPES other than DES was responsible for this effect.
Previous in vivo and in vitro studies have shown that PC-SPES exhibits estrogenic activity.
Clinical studies have noted significant estrogen-like effects when 3 to 9 320-mg capsules are
administered per day (Small et al., 2000; de la Taille et al., 2000). These effects include loss of
libido and sexual potency, gynecomastia and thromboembolism. Di Paola et al. (DiPaola et al.,
1998) showed that PC-SPES had estrogenic activity by using transcription activation assays in
yeast and uterotrophic assays in ovarectamized mice. Our present results, suggesting that the
estrogenic activity of PC-SPES is variable and dependent on the particular lot examined agrees
with the recent report by Sovak et al. (Sovak et al., 2002), which showed that the composition of
PC-SPES varies by lot. Chemical analyses detected various amounts of synthetic drugs, including
18 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 DES as well as several natural products, including the licochalcone A.
One explanation for the estrogenic activity of PC-SPES that is consistent with our results is the presence of DES contamination. However, it has also been suggested that this activity is chemically and functionally distinct from DES. For instance, DiPaola et al. (DiPaola et al., 1998) detected an unknown organic substance that co-eluted with DES on HPLC, but did not match the retention time of DES, estrone, or estradiol when fractioned with GC/MS. Phytochemical Downloaded from components of PC-SPES, such as licorice root and its derivative licochalcone-A exhibit phytoestrogenic activity (Rafi et al., 2000). LNCaP cells, an androgen-responsive human prostate cancer cell line, exhibits a differential proliferative response to PC-SPES compared to estradiol jpet.aspetjournals.org and different gene expression profile compared to diethylstilbestrol (Hsieh et al., 2002; Bonham et al., 2002). Although our HPLC is in agreement with GC/MS and NMR data from other at ASPET Journals on September 26, 2021 laboratories (California Department of Health, 2002; Sovak et al., 2002), suggesting that PC-SPES lot #5430125 contains DES while lot #5431249 does not, it is possible that other phytochemical components, acting alone or in synergy with DES, contribute to the profile of estrogenic activity that was observed. Furthermore, the chemical and functional differences that we observed between lots of PC-SPES highlight the problems that arise due to variation in concentration of active ingredients when an herbal preparation is not rigorously standardized and its production is insufficiently monitored.
The statistically significant difference in T levels and relative weights of the ventral prostate and seminal vesicles in male rats treated with DES compared to rats treated with PC-
SPES lot #5430125 may be due to a difference in the dose. While animals treated with DES were given a dose of 37.5 µg/kg, animals treated with 250 mg/kg PC-SPES lot #5430125 theoretically
19 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 received 20 µg/kg DES (based on an estimated contamination of 25 µg/320 mg capsule). If in addition to DES, lot #5430125 contains higher levels of phytoestrogen, the differential effects could also be attributed to differences in potency between this phytoestrogen and DES. DES treatment did not have a greater effect on relative uterine weight than PC-SPES lot #5430125.
Doses of PC-SPES used in our studies were calculated using an allometric exponent of
0.75 (based on whole body oxygen consumption) (Porter, 2001) and agree with the previous work Downloaded from of Kubota et al. (Kubota et al., 2000). PC-SPES doses of 50 mg/kg/day and 250 mg/kg/day in a
200 mg rat translates to approximately 3 and 13 capsules of PC-SPES per day respectively for a 70 jpet.aspetjournals.org kg man. A rat dose of 37.5 µg/kg DES is equivalent to half the therapeutic dose of DES prescribed for human prostate cancer and reflects a dose of approximately 24 capsules of PC-
SPES lot#5430125. These doses of PC-SPES, although higher than those used in clinical trials, at ASPET Journals on September 26, 2021 are relevant given the fact that PC-SPES is not purchased with a prescription and dose and duration of treatment could therefore be extremely variable.
Although the two lots of PC-SPES exhibited different estrogenic activities, both lots had similar effects on hepatic CYP enzymes. Both lots of PC-SPES and DES reduced total hepatic
CYP3A2 activity and protein levels. While it appeared that there was a trend towards inhibition of
CYP3A2 mRNA by both lots of PC-SPES, the effect was not statistically significant. We are therefore not able to confirm whether the inhibition of CYP3A protein by PC-SPES and DES occurred at a transcriptional or post-transcriptional level. To the best of our knowledge, this is the first time that inhibition of CYP3A2 activity by DES has been reported. While neither lot of PC-
SPES or DES alone significantly affected protein levels of CYP2C11, DES significantly inhibited
CYP2C11-dependent T hydroxylation.
20 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 The inhibitory effect of DES on CYP3A2 and CYP2C11 is consistent with an alteration of
the hypothalamic-pituitary axis. Both enzymes are expressed in a male specific manner and the
sexually dimorphic pattern of growth hormone secretion is thought to play a key role in the
regulation of these enzymes (Morgan et al., 1985; Waxman et al., 1995). Both androgens and
estrogens can influence growth hormone secretion (Hull and Harvey, 2002). Although GC/MS
data suggests that lot #5730125 contains DES, this lot did not exert the same effects on CYP2C11 Downloaded from as DES alone. This apparent inconsistency may be due to the lower dose of DES in PC-SPES lot
#5430125 when compared to the dose of DES received by rats treated with DES alone.
Because the response of CYP3A to PC-SPES was not dependent on whether it contained jpet.aspetjournals.org
DES, a phytochemical component of PC-SPES other than DES is most likely responsible for the
inhibitory effect on CYP3A2. PC-SPES is a complex mixture containing the concentrated extracts at ASPET Journals on September 26, 2021 from 8 different herbs. It is comprised of perhaps hundreds of distinct compounds, several of
which may contribute to CYP3A2 inhibition. Baicalein and wogonin, major chemical constituents
of Scutellaria baicalensis, Georgi, are flavone derivatives that have been shown to decrease
hepatic CYP3A protein and activity in vivo (Ueng et al., 2000). Panax ginseng contains saponin
constituents (panaxosides and gisenosides) (Darzynkiewicz et al., 2000). Purified Ginsenosides
display weak inhibitory activity against recombinant human CYP3A4 (Henderson et al., 1999).
Glycyrrhiza contains the phytocoumarins glycyrol, glycyrin and glycycoumarin (Tanaka et al.,
2001). Studies suggest that coumarin and furanocoumarins exhibit inhibitory effects on the in
vitro activity of CYP3A in human microsomes (Bailey et al., 1998; Guo et al., 2000). While many
phytochemicals can inhibit the activity of CYP3A in a reversible manner, the changes observed
with PC-SPES and DES appear to represent changes in the level of the enzyme expression. It is
21 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 also possible that a component of PC-SPES acts as a suicide inhibitor of the enzyme. This could lead to enhanced degradation of the enzyme, similar to what has been reported for the enzyme inactivated by heme modification with cumene hydroperoxide (Korsmeyer et al., 1999) or mechanism-based inactivators such as 3,5-dicarboethyoxy-2,6-dimethyl-4-ethyl-1,4- dhydropyridine (DDEP) (Wang et al., 1999).
CYP3A is the largest subfamily of CYP enzymes expressed in the human liver and Downloaded from gastrointestinal tract. It is involved in the metabolism of endogenous substrates such as sex steroids, bile acids and retinoic acids as well as clinically important drugs such as the anticancer jpet.aspetjournals.org drug Taxol, the 5-α reductase inhibitor finasteride, and the antianxiety agents midazolam and traizolam. Inhibition of CYP3A therefore has important implications for potential herbal-drug interactions (Thummel and Wilkinson, 1998). Thus, if common regulatory mechanisms exist in at ASPET Journals on September 26, 2021 humans, PC-SPES consumption by prostate cancer patients could potentially cause increased bioavailability of co-administered medications. This could be readily examined in vivo using noninvasive assessments of CYP3A4 activity such as the erythromycin breath test (Watkins, 1994) or midazolam clearance (Lin et al., 2001).
In summary, although herbal dietary supplements are often considered by patients to be safe and free from side effects, these studies demonstrate that in addition to the potential risk of interaction between herbal medicines and conventional drugs, the possibility for inconsistent composition, contamination or product adulteration with prescription drugs is possible. Herbal preparations such as PC-SPES need to undergo rigorous scientific evaluation to assess therapeutic value, safety, purity, and potential for herb-drug interactions.
Acknowledgments
22 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 We thank Lisa Bleyle and Henry Stadelman for their expert technical assistance. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
23 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
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JPET #48645 Footnotes
Supported by NIH grant R21 AT000670 (CER) and the Reproductive Biology Training Grant at
Oregon Health & Science University NICHD T32 HD07133. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
30 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 Fig 1. Comparison of HPLC Profiles of PC-SPES. HPLC was employed to characterize the
composition of 70% ethanolic extracts of PC-SPES lot #5430125 (A) and lot#5431249 (B).
Although the retention times of most absorbance peaks are similar, each lot exhibits a unique
HPLC profile and different constituent ratios. Arrows point to a peak at 38 minutes that co-elutes with warfarin, and to a unique peak at 39 minutes in lot #5430125 that co-elutes with DES.
Fig 2. Effects of PC-SPES and DES on CYP3A and CYP 2C11-dependent activities. T-6β- Downloaded from
hydroxylase activity (A) and T-16α-hydroxylase activity (B) were determined in hepatic
microsomes from male rats treated with vehicle (n=7) vs PC-SPES lot #5430125 (50 mg/kg, n=7; jpet.aspetjournals.org 250 mg/kg, n=7); vehicle (n=6) vs PC-SPES lot #5431249 (250 mg/kg, n=6); or vehicle (n=7) vs
DES (37.5 µg/kg, n=7). Graphs show % control T-6β-OH and T-16α-OH (mean " SE, *
P<0.05). Both lots of PC-SPES at a dose of 250 mg/kg decreased CYP3A-dependent testosterone at ASPET Journals on September 26, 2021 T-6β-hydroxylation. DES at a dose of 37.5 µg/kg significantly decreased CYP2C11-dependent T-
16α-hydroxylation.
Fig 3. Effects of both lots of PC-SPES and DES on CYP3A and CYP2C11protein levels.
Hepatic microsomes were examined for levels of CYP3A and CYP2C11 protein by western blot
analysis as described in Materials and Methods. Quantitation of western blots and representative
images are shown for A. rats treated with vehicle (n=7), PC-SPES lot #5430125 (50 mg/kg (n=7)
or PC-SPES lot #5430125 250 mg/kg (n=7); B. rats treated with vehicle (n=6) or PC-SPES lot
#5431249 (250 mg/kg, n=6); C. rats treated with vehicle (n=7) or DES (37.5 µg/kg, n=7). Graphs
show % control CYP protein expression (bars, SE, * P<0.05). Both lots of PC-SPES at a dose of
250 mg/kg and DES at a dose of 37.5 µg/kg significantly decreased CYP3A protein levels. None
31 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #48645 of the treatments significantly altered levels of CYP2C11 protein.
Fig 4: Effects of PC-SPES and DES on hepatic CYP3A gene expression. Total RNA was extracted from rat livers and northern blotting was performed as described in Materials and
Methods. A. Relative CYP3A2 gene expression in male rats treated with vehicle (n=7) or PC-
SPES lot #5430125 (250 mg/kg, n=7); vehicle (n=6) or PC-SPES lot #5431249 (250 mg/kg, n=6); and vehicle (n=7) or DES (37.5 µg/kg, n=7). B. Relative CYP3A18 gene expression in male rats Downloaded from treated with vehicle (n=7) or PC-SPES lot #5430125 (250 mg/kg, n=7); and vehicle (n=6) or PC-
SPES lot #5431249 (250 mg/kg, n=6). Graphs represent quantitation (bars, SE) of relative jpet.aspetjournals.org CYP3A mRNA levels normalized to 18S rRNA levels. All treatments showed a trend towards inhibition of CYP3A2 gene expression when compared with vehicle alone, but the effects were not statistically significant. at ASPET Journals on September 26, 2021
32 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 Table 1: Effect of orally administered PC-SPES and DES on organ & body weights of male rats (mean ± S.E.)
Organ Weight/Body Weight (mg/g) This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
Treatment BW V.Pros SV LA Apit Adrenal Kidney Spleen JPET FastForward.PublishedonApril3,2003asDOI:10.1124/jpet.102.048645
Cont (n=7) 253 ± 14 1.16 ± 0.08a 1.38 ± 0.10a,b 0.52 ± 0.04 0.037 ± .003 0.24 ± 0.02 3.75 ± 0.16a 2.14 ± 0.11
#5430125 (50 mg/kg, n=7) 257 ± 11 1.01 ± 0.06a 1.18 ± 0.14b,c 0.53 ± 0.03 0.040 ± .004 0.22 ± 0.03 4.09 ± 0.06a,b 2.41 ± 0.11
#5430125 (250 mg/kg, n=7) 249 ± 4 0.83 ± 0.04b 0.96 ± 0.06c 0.40 ± 0.05 0.039 ± .003 0.21 ± 0.02 4.18 ± 0.08b 2.15 ± 0.06
DES (37.5 µg/kg, n=7) 262 ± 13 0.50 ± 0.04c 0.64 ± 0.05d 0.41 ± 0.02 0.038 ±.001 0.22 ± 0.01 4.38 ± 0.16b 2.44 ± 0.06
Cont (n=6) 314 ± 7a 1.09 ± 0.08 1.41 ± 0.10 0.65 ± 0.05 0.030 ± .001 0.16 ± 0.01 7.45 ± 0.49 2.32 ± 0.07a
#5431249 (250 mg/kg, n=7) 270 ± 6b 1.02 ± 0.05 1.32 ± 0.06 0.64 ± 0.02 0.031 ± .005 0.17 ± 0.01 8.42 ± 0.12 1.83 ± 0.10b
Data was analyzed by either t-tests (comparison between two groups) or by one-way ANOVA followed by Fisher's least square difference (comparisons between multiple groups). Means with different superscripts are significantly different, P < 0.05
Abbreviations: body weight (BW); ventral prostate (V.Pros); seminal vesicle (SV); levator ani (LA); anterior pituitary (Apit)
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
Table 2: Effect of PC-SPES and DES on gonadal steroids and gonadotropin hormones in male rats (mean ± S.E.)
Hormone
Treatment T (ng/ml) DHT (ng/ml) LH (ng/ml)
Cont (n=7) 4.0 ± 1.2a 0.6 ± 0.2a,c 0.8 ± 0.2a
#5430125 (50 mg/kg, n=7) 2.5 ± 0.9a 0.3 ± 0.1a 0.5 ± 0.1b
#5430125 (250 mg/kg, n=7) 1.4 ± 0.4a,b 0.2 ± 0.1a 0.2 ± 0.02c Downloaded from
DES (37.5 µg/kg, n=7) 0.7 ± 0.4b 0.9 ± 0.03b 0.5 ± 0.06a,b
jpet.aspetjournals.org Cont (n=6) 4.2 ± 0.9 0.9 ± 0.3 1.0 ± 0.1
#5431249 (250 mg/kg, n=6) 3.3 ± 0.7 1.1 ± 0.4 0.7 ± 0.2
Data were analyzed by either t-tests (comparisons between two groups) or by one-way ANOVA at ASPET Journals on September 26, 2021 followed by Fisher's least square difference (comparisons between multiple groups). Means with different superscripts are significantly different, P < 0.05 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
Table 3. Effect of PC-SPES and DES on uterine weights (mean ± S.E.)
Treatment BW(g) Uterine wt/BW (g/kg)
Cont (n=4) 233.5 ± 10.4a 0.37 ± 0.09a
#5430125 (250 mg/kg, n=5) 178.0 ± 6.2b 1.1 ± 0.1b
DES (37.5 µg/kg, n=5) 200.0 ± 5.3c 1.1 ± 0.06b
Control (n=6) 253.0 ± 1.7a 0.52 ± 0.03a Downloaded from
#5431249 (250 mg/kg, n=6) 196.0 ± 9.8b 0.69 ± 0.07a
DES (37.5 µg/kg, n=6) ± b ± b
198.0 2.8 1.7 0.12 jpet.aspetjournals.org
Data were analyzed by one-way ANOVA followed by Fisher's least square difference. Means with different superscripts are significantly different, P < 0.05 at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 3, 2003 as DOI: 10.1124/jpet.102.048645 This article has not been copyedited and formatted. The final version may differ from this version.
Table 4. Oligonucleotide probes for Rat CYP3A mRNA and 18S rRNA
Target Probe
CYP3A2 5’ CATCTCCTTGAGTCTTCCAC 3’
CYP3A18 5' AGCCACTGAGCCTGTTCAGTGA 3'
CYP3A9 5' CTTGCACAGAGGCTGAGAAAGC 3'
18S 5' CACCTCTAGCGCCGCAATAC 3' Downloaded from
jpet.aspetjournals.org at ASPET Journals on September 26, 2021