The nutritional herb Epimedium grandiflorum inhibits the growth in a model for the Luminal A molecular subtype of breast cancer.

The Luminal A subtype of breast cancer expresses the estrogen receptor (ER)-α and progesterone receptor (PR), but not the human epidermal growth factor receptor (HER)-2 oncogene. This subtype of breast cancer responds to endocrine therapy involving the use of selective estrogen receptor modulators and/or inhibitors of estrogen biosynthesis. However, these therapeutic agents are frequently associated with long-term systemic toxicity and acquired tumor resistance, emphasizing the need to identify non-toxic alternative treatments for chemo-endocrine therapy responsive breast cancer. The present study utilized the human mammary carcinoma-derived, ER+/PR+/HER-2- MCF-7 cell line as a model of the Luminal A subtype of breast cancer to examine the growth inhibitory effect of the Chinese nutritional herb Epimedium grandiflorum (EG) and determine the mechanisms underlying this effect. MCF-7 cells maintained in a serum-depleted culture medium retained their ability to grow in response to 17ß-estradiol (E2). Treatment of the MCF-7 cells with EG resulted in dose-dependent inhibition of E2-promoted growth. Mechanistically, EG inhibited E2-promoted cell cycle progression through G1 stage arrest and modulated the cellular metabolism of E2, increasing the formation of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Long-term treatment of MCF-7 cells with EG inhibited E2-promoted anchorage independent growth, a surrogate in vitro biomarker of tumorigenesis. In conclusion, the results of the present study demonstrate the growth inhibitory effects of EG on MCF-7 cells and identified clinically relevant mechanistic leads for its anti-tumorigenic efficacy.

Inhibitory effects of Chinese nutritional herbs in isogenic breast carcinoma cells with modulated estrogen receptor function.

In estrogen receptor (ER)+ MCF-7 cells, ER represents a ligand-activated transcription factor, and 17ß-estradiol (E2) represents its physiological ligand. Maintenance of the human breast carcinoma-derived MCF-7 cells with 0.7% serum selected a proliferative sub-population of E2-responsive cells with transiently non-functional ER due to limited availability of E2. Culture of MCF-7 cells in the presence of either 0.7% serum, <1 nM E2 or 0.7% serum + 20 nM E2 selected isogenic cells with either non-functional ER (ER-NF) or functional ER (ER-F) phenotype. The two phenotypes responded to the growth-promoting effects of E2 and to the growth-inhibitory effects of the selective ER modulator tamoxifen, indicating retention of E2 responsiveness. Comparative dose-response experiments with Chinese nutritional herbs on ER-NF and ER-F cells identified the inhibitory concentration (IC)50 values for these herbs, while the IC50 ratios for the ER-NF:ER-F phenotypes facilitated their rank ordering in terms of efficacy. Out of the 11 efficacious herbs tested, five herbs exhibited ER-F > ER-NF inhibitory activity, four exhibited ER-F = ER-NF inhibitory activity and two exhibited ER-NF > ER-F inhibitory activity. Extracts from representative herbs, Lycium barbarum bark, Epimedium grandiflorum and Cornus officinalis, from each of the three groups inhibited anchorage-independent growth, induced G1 or G2/M arrest and/or apoptosis, and generated anti-proliferative E2 metabolites. The differential growth inhibition in ER-NF and ER-F phenotypes, together with the mechanistic efficacy of representative herbs, identified potential leads for their efficacy on ER+ and/or ER- breast cancer.

The effect of oral 3,3'-diindolylmethane supplementation on the 2:16α-OHE ratio in BRCA1 mutation carriers.

Hormonal exposures are known to influence breast cancer risk among women with a BRCA1 mutation. Thus, dietary factors that increase the 2-hydroxyestrone (OHE):16α-OHE ratio, a biomarker inversely related to breast cancer development, may also influence cancer risk. We conducted a dietary intervention study to evaluate the ability of 300 mg/day of 3,3'-diindolylmethane (DIM) to increase the urinary 2:16α-OHE ratio in 20 women with a BRCA1 mutation. BRCA1 mutation carriers (n = 15) were assigned to receive 300 mg/day of Rx Balance BioREsponse DIM for 4-6 weeks (intervention group) and five BRCA1 mutation carriers did not take DIM (control group). The urinary 2:16α-OHE ratio was assessed at baseline and after 4-6 weeks by immunoassay. There was no significant effect of DIM on the 2:16α-OHE ratio (2.4 at baseline vs. 3.0 after the intervention, P = 0.35). A short dietary intervention with DIM did not significantly increase the 2:16α-OHE ratio in female BRCA1 mutation carriers. Larger studies investigating the effect of dietary or lifestyle interventions on circulating hormone levels in these high-risk women are warranted.

Comparative efficacy of extracts from Lycium barbarum bark and fruit on estrogen receptor positive human mammary carcinoma MCF-7 cells.

Chemo-endocrine therapy for estrogen receptor positive (ER(+)) breast cancer exhibits acquired tumor resistance. Herbal medicines provide integrative support for breast cancer patients. Present study compared the efficacy of aqueous extracts from Lycium barbarum bark (LBB) and Lycium barbarum fruit (LBF) on ER(+) MCF-7 cells. Cellular growth and 17ß-estradiol (E2) metabolism quantified the efficacy. MCF-7 cells maintained in serum depleted medium+ E2 exhibited increased anchorage-dependent and anchorage-independent growth. LBB exhibited greater potency than LBF (95% reduction in IC50). LBB produced a 6.8-fold increase, 40% decrease, and a 3.7-fold increase in 2-hydroxyestrone (2-OHE1), 16α-hydroxyestrone (16α-OHE1), and estriol (E3) formation. The corresponding values for LBF were 3.9, 33, and 10.5. LBB produced a16.3-fold and a twofold increase in 2-OHE1:16α-OHE1 and E3:16α-OHE1 ratios, whereas LBF produced a sixfold and a 2.9-fold increase. The efficacy of LBB is due to increased 2-OHE1 formation, whereas that of LBF is due to accelerated conversion of 16α-OHE1 to E3. Specific growth inhibitory profiles of LBB and LBF may be due to their distinct chemical composition and their complementary actions on E2 metabolism. This study validates a mechanistic approach to identify efficacious herbal extracts for clinical ER(+) breast cancer.

Evaluation of 3,3'-diindolylmethane with gardasil quadrivalent HPV vaccine in K14-HPV16-transgenic mice cervical histology.

BACKGROUND: The effects of 3,3'-diindolylmethane (DIM) together with the Gardasil vaccine on cervical histology were evaluated using the K14-HPV16-transgenic mouse model. The possibility that DIM could enhance the efficacy of this preventive vaccine in this model was explored. MATERIALS AND METHODS: Transgenic mice were given 1000 mg/kg of DIM in the diet for 28 weeks. The mice were injected with Gardasil Quadrivalent HPV vaccine. Some mice were sacrificed at 28 weeks. Other groups were removed from the DIM diet after 28 weeks to a diet with no DIM for either 4 or 8 weeks. RESULTS: Cervical histology indicated that a high percentage of transgenic mice fed DIM and vaccinated with Gardasil manifested normal cervical epitheliums at 4 weeks after DIM discontinuation. CONCLUSION: Vaccination pre-supplemented with DIM may provide with a window of protection of at least four weeks in this transgenic model. However, extrapolation to the effect in humans is beyond the limited scope of the histological data presented here.

Polychlorinated biphenyl exposure, diabetes and endogenous hormones: a cross-sectional study in men previously employed at a capacitor manufacturing plant.

BACKGROUND: Studies have shown associations of diabetes and endogenous hormones with exposure to a wide variety of organochlorines. We have previously reported positive associations of polychlorinated biphenyls (PCBs) and inverse associations of selected steroid hormones with diabetes in postmenopausal women previously employed in a capacitor manufacturing plant. METHODS: This paper examines associations of PCBs with diabetes and endogenous hormones in 63 men previously employed at the same plant who in 1996 underwent surveys of their exposure and medical history and collection of bloods and urine for measurements of PCBs, lipids, liver function, hematologic markers and endogenous hormones. RESULTS: PCB exposure was positively associated with diabetes and age and inversely associated with thyroid stimulating hormone and triiodothyronine-uptake. History of diabetes was significantly related to total PCBs and all PCB functional groupings, but not to quarters worked and job score, after control for potential confounders. None of the exposures were related to insulin resistance (HOMA-IR) in non-diabetic men. CONCLUSIONS: Associations of PCBs with specific endogenous hormones differ in some respects from previous findings in postmenopausal women employed at the capacitor plant. Results from this study, however, do confirm previous reports relating PCB exposure to diabetes and suggest that these associations are not mediated by measured endogenous hormones.

3,3'-Diindolylmethane increases serum interferon-γ levels in the K14-HPV16 transgenic mouse model for cervical cancer.

UNLABELLED: While cervical cancer incidence and mortality rates have declined in the United States, this cancer represents a worldwide threat. Human papilloma viral infection causes cervical neoplasia (CIN). 3,3'-Diindolylmethane (DIM) prevents or inhibits the progression from cervical dysplasia to cancer. The aim of this study is to determine the most effective dose of DIM given continuously in food, that significantly increases serum interferon gamma levels (IFN-γ) in the K14-HPV16 transgenic mouse model for cervical cancer. MATERIALS AND METHODS: Five doses of DIM in food were administered to the mouse model for 20 weeks. Serum Interferon gamma (IFN-γ) levels and estrogen metabolite levels were quantified. RESULTS: At 1000 ppm DIM, serum IFN-γ concentrations were significantly increased (p<0.0396). The estrogen metabolites were unchanged. IFN-γ concentrations in CIN free mice and the percentage of CIN free transgenic mice were well correlated (r=0.88). DISCUSSION: Significant increases in IFN-γ serum concentrations that correlate with the percentage of CIN free mice in each group indicate that 1000 ppm of DIM in food may be the most effective dose for future studies. These results may eventually lead to new and effective vaccination strategies in women already infected with the human papilloma virus.

Adipocyte-derived factor as a modulator of oxidative estrogen metabolism: implications for obesity and estrogen-dependent breast cancer.

The role of body fat as a risk factor for breast cancer has been well established. A decrease in the urinary 2/16α-hydroxyestrone ratio has also been shown to be a risk marker for breast cancer. These two observations are connected by the fact that obese women have decreased levels of 2-hydroxyestrone. To test the hypothesis that fat depots secrete factors that inhibit 2-hydroxylation, the effect of substances released into the media from adipocytes incubated in Krebs-Ringer buffer, on estrogen metabolism by MCF-7 cells in minimum essential medium eagle (MEM) plus adipocyte-conditioned media (ACM) was studied. The 1:1 ACM-MEM culture system resulted in a substantial and highly significant decrease in 2-hydroxylation of estradiol. This inhibition was partially reversed by the addition of indole-3-carbinol, a potent inducer of 2-hydroxylation of estradiol. Centrifugal sizing showed that the active 2-hydroxylation inhibitor in the medium had a molecular weight of about 30 kDa. These results suggest a mechanism for the decrease in 2-hydroxylation of estradiol that is observed in obese women and the increase in 2-hydroxylation observed in women with depleted fat depots.

Associations of polychlorinated biphenyl exposure and endogenous hormones with diabetes in post-menopausal women previously employed at a capacitor manufacturing plant.

There is an increasing body of literature showing associations of organochlorine exposure with risk of diabetes and insulin resistance. Some studies suggest that associations differ by gender and that diabetes risk, in turn, may be affected by endogenous steroid hormones. This report examines the relationships of serum PCBs and endogenous hormones with history of diabetes in a cohort of persons previously employed at a capacitor manufacturing plant. A total of 118 women were post-menopausal with complete data, of whom 93 were not using steroid hormones in 1996, at the time of examination, which included a survey of exposure and medical history, height, weight and collection of blood and urine for measurements of lipids, liver function, hematologic markers and endogenous hormones. This analysis examines relationships of serum polychlorinated biphenyls (PCBs), work exposure and endogenous hormones with self-reported history of diabetes after control for potential confounders. All PCB exposure groups were significantly related to history of diabetes, but not to insulin resistance as measured by the homeostatic model assessment of insulin resistance (HOMA-IR) in non-diabetics. Diabetes was also independently and inversely associated with follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS) and triiodothyronine (T3) uptake. HOMA-IR was positively associated with body mass index (BMI) and C-reactive protein (CRP) and inversely associated with sex hormone binding globulin (SHBG) and T3 uptake after control for PCB exposure. Possible biologic mechanisms are discussed. This study confirms previous reports relating PCB exposure to diabetes and suggests possible hormonal pathways deserving further exploration.

Results from a dose-response study using 3,3'-diindolylmethane in the K14-HPV16 transgenic mouse model: cervical histology.

The human papilloma virus is the major cause of cervical cancer. Viral infection initiates cervical intraepithelial neoplasia, which progresses through several stages to cervical cancer. The objective of this study is to identify the minimum effective dose of diindolylmethane that prevents the progression from cervical dysplasia to carcinoma in situ. We document cervical histology in K14-HPV16 mice receiving different doses of diindolylmethane. Urinary diindolylmethane concentrations are reported. Diindolylmethane could enhance the efficacy of human papilloma virus vaccines, creating a new therapeutic use for these vaccines in women already infected with the virus. Five doses (0-2,500 ppm) of diindolylmethane were incorporated into each mouse diet. The reproductive tract was serially sectioned and urine was obtained for analysis of urinary diindolylmethane. The results indicate that 62% of mice receiving 1,000 ppm diindolylmethane remained dysplasia-free after 20 weeks compared with 16% of mice receiving no diindolylmethane and 18% receiving 500 ppm; 1,000 ppm of 3,3'-diindolylmethane in the diet completely suppressed the development of cervical cancer. Urinary diindolylmethane levels increased significantly as diindolylmethane in food increased. These findings imply usefulness for diindolylmethane in the search to prevent cervical cancer when used in combination with prophylactic or therapeutic vaccines.

3,3'-diindolylmethane modulates estrogen metabolism in patients with thyroid proliferative disease: a pilot study.

BACKGROUND: The incidence of thyroid cancer is four to five times higher in women than in men, suggesting a role for estrogen (E2) in the pathogenesis of thyroid proliferative disease (TPD) that comprises cancer and goiter. The objective of this study was to investigate the antiestrogenic activity of 3,3'-diindolylmethane (DIM), a bioactive compound derived from cruciferous vegetables, in patients with TPD. METHODS: In this limited phase I clinical trial study, patients found to have TPD were administered 300 mg of DIM per day for 14 days. Patients subsequently underwent a total or partial thyroidectomy, and tissue, urine, and serum samples were collected. Pre- and post-DIM serum and urine samples were analyzed for DIM levels as well as estrogen metabolites. DIM levels were also determined in thyroid tissue samples. RESULTS: DIM was detectable in thyroid tissue, serum, and urine of patients after 14 days of supplementation. Urine analyses revealed that DIM modulated estrogen metabolism in patients with TPD. There was an increase in the ratio of 2-hydroxyestrones (C-2) to 16α-hydroxyestrone (C-16), consistent with antiestrogenic activity that results in more of C-2 product compared with C-16. CONCLUSION: Our data suggest that DIM enhances estrogen metabolism in TPD patients and can potentially serve as an antiestrogenic dietary supplement to help reduce the risk of developing TPD. The fact that DIM is detected in thyroid tissue implicates that it can manifest its antiestrogenic activity in situ to modulate TPD.

Effects of a breast-health herbal formula supplement on estrogen metabolism in pre- and post-menopausal women not taking hormonal contraceptives or supplements: a randomized controlled trial.

INTRODUCTION: Both indole-3-carbinol and dietary lignans have beneficial effects on estrogen metabolism and breast cancer risk. There is no published literature on the effects of a combination product. This study was designed to investigate the impact of a combination product on estrogen metabolism. The major trial objective was to determine whether a breast health supplement containing indole-3-carbinol and hydroxymatairesinol lignan would alter estrogen metabolism to favour C-2 hydroxylation and reduce C-16 hydroxylation. Higher concentrations of C-2 metabolites and lower concentrations of C-16 metabolites may reduce breast cancer risk and risk for other hormonally-related cancers. METHODS: Forty-seven pre-menopausal and forty-nine post-menopausal women were recruited for this study, and were divided by random allocation into treatment and placebo group. The treatment supplement contained HMR lignan, indole-3-carbinol, calcium glucarate, milk thistle, Schisandra chinesis and stinging nettle, and each woman consumed either treatment or placebo for 28 days. At day 0 and day 28, blood samples were analysed for serum enterolactone concentrations, and first morning random urine samples were assessed for estrogen metabolites. Repeated measures ANOVA statistical testing was performed. RESULTS: In pre-menopausal women, treatment supplementation resulted in a significant increase (P < 0.05) in urinary 2-OHE concentrations and in the 2:16α-OHE ratio. In post-menopausal women, treatment supplementation resulted in a significant increase in urinary 2-OHE concentrations. In pre- and post-menopausal women combined, treatment supplementation produced a significant increase in urinary 2-OHE concentration and a trend (P = 0.074) toward an increased 2:16α-OHE ratio. There were no significant increases in serum enterolactone concentrations in the treatment or placebo groups. CONCLUSIONS: Supplementation with a mixture of indole-3-carbinol and HMR lignan in women significantly increased estrogen C-2 hydroxylation. This may constitute a mechanism for the reduction of breast cancer risk as well as risk for other estrogen-related cancers. Further studies with higher numbers of subjects are indicated.

Diindolylmethane inhibits cervical dysplasia, alters estrogen metabolism, and enhances immune response in the K14-HPV16 transgenic mouse model.

This study was designed to establish whether 3,3'-diindolylmethane (DIM) can inhibit cervical lesions, alter estrogen metabolism in favor of C-2 hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse model. Mice were bred, genotyped, implanted with E(2) pellets (0.25 mg/90-day release) under anesthesia, and divided into groups. Wild-type and transgenic mice were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and reproductive tracts were obtained. Blood was analyzed for estrogen metabolites and IFN-gamma. The cervical transformation zone was sectioned and stained for histology. Estradiol C-2 hydroxylation and serum IFN-gamma levels were significantly increased over controls in wild-type and transgenic mice receiving DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was observed. Wild-type mice fed DIM displayed a normal thin epithelium. In transgenic mice without DIM, epithelial cell projections into the stroma (papillae) were present. An additional degree of nuclear anaplasia in the stratum espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM displayed some mild hyperplasia of the squamous epithelium. DIM increases estrogen C-2 hydroxylation in this model. Serum INF-gamma was increased, indicating increased immune response in the DIM-fed animals. Histopathology showed a marked decrease in cervical dsyplasia in both wild-type and transgenic mice, indicating that DIM delays or inhibits the progression from cervical dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have shown that DIM inhibits the development of E6/E7 oncogene-induced cervical lesions.

Lycium barbarum inhibits growth of estrogen receptor positive human breast cancer cells by favorably altering estradiol metabolism.

Selective estrogen receptor modulators represent accepted therapy for estrogen receptor positive (ER+) breast cancer, exhibit adverse side effects, and reduce patient compliance. The use of phytoestrogen containing herbal medicines is limited because of efficacy and safety concerns. The ER+ MCF-7 model examined growth inhibitory effects of the medicinal herb Lycium barbarum (LB) and identified mechanistic leads for its efficacy. The MCF-7 cells maintained in 0.7% serum (17beta-estradiol, E2 < 1 nM) exhibited 11%-87% increased growth after treatment with 1nM to 20 nM E2. Growth promotion with 20 nM E2 exhibited 5.2-fold increased estrone (E1), 35.7% increased 2-hydroxyestrone (2-OHE1), 15.4% increased 16alpha-hydroxyestrone (16alpha-OHE1), and eightfold increased estriol (E3) formation. Treatment of E2 stimulated cells with LB exhibited a dose-dependent growth inhibition of 9.5%-42.8% at Day 3 and 33.9%-83.9% at Day 7. The 3-day inhibitory response to 1% LB (maximum cytostatic concentration) exhibited 84.8% increased E1, 3.6-fold increased 2-OHE1, 33.3% decreased 16alpha-OHE1, and 9.2-fold increased E3 formation. Thus, MCF-7 cells retain their mitogenic and metabolic response to E2 and LB downregulates E2-stimulated growth via the formation of antiproliferative 2-OHE1 and accelerated conversion of mitogenic 16alpha-OHE1 to antimitogenic E3.

Estrogen hydroxylation--the good and the bad.

Although estradiol itself is primarily responsible for female development, the metabolites are responsible for many of the other positive and negative properties of estrogens. Phase I metabolism of estradiol is exclusively oxidative unlike the other steroid hormones and involves a series of hydroxylations. The specific hydroxylations can be induced or suppressed by endogenous or exogenous compounds that influence the cytochrome enzymes that act on specific sites on the molecule. Modulation of estrogen hydroxylation is essential since some of the other metabolites increase the risk of breast and other hormone-related cancers. The various hydroxylation pathways are discussed as well as the effects of the products of estrogen hydroxylation. The interaction between the human papilloma virus (HPV) and 16alpha-hydroxyestrone is discussed with reference to recurrent respiratory papillomatosis, cervical dysplasia, and cervical cancer. The role of estrogen metabolites in predicting the relative risk for breast cancer is evaluated using prospective and case-control studies. In one pilot study a factor that is a component of body fat is identified to be an inhibitor of estrogen C-2 hydroxylation. The role of environmental toxins like the phthalate esters and how these compounds increase risk for hormonal cancers is examined in a second pilot study.

Dietary seaweed modifies estrogen and phytoestrogen metabolism in healthy postmenopausal women.

Seaweed and soy foods are consumed daily in Japan, where breast cancer rates for postmenopausal women are significantly lower than in the West. Likely mechanisms include differences in diet, especially soy consumption, and estrogen metabolism. Fifteen healthy postmenopausal women participated in this double-blind trial of seaweed supplementation with soy challenge. Participants were randomized to 7 wk of either 5 g/d seaweed (Alaria) or placebo (maltodextrin). During wk 7, participants also consumed a daily soy protein isolate (2 mg isoflavones/kg body weight). After a 3-wk washout period, participants were crossed over to the alternate supplement schedule. There was an inverse correlation between seaweed dose (mg/kg body weight) and serum estradiol (E2) (seaweed-placebo = y = -2.29 x dose + 172.3; r = -0.70; P = 0.003), [corrected] which was linear across the range of weights. Soy supplementation increased urinary daidzein, glycitein, genistein, and O-desmethylangolensin (P = 0.0001) and decreased matairesinol and enterolactone (P < 0.05). Soy and seaweed plus soy (SeaSoy) increased urinary excretion of 2-hydroxyestrogen (2-OHE) (P = 0.0001) and the ratio of 2-OHE:16alpha-hydroxyestrone (16alphaOHE(1)) (P = 0.01). For the 5 equol excretors, soy increased urinary equol excretion (P = 0.0001); the combination of SeaSoy further increased equol excretion by 58% (P = 0.0001). Equol producers also had a 315% increase in 2:16 ratio (P = 0.001) with SeaSoy. Seaweed favorably alters estrogen and phytoestrogen metabolism and these changes likely include modulation of colonic bacteria.

3,3'-Diindolylmethane and genistein decrease the adverse effects of estrogen in LNCaP and PC-3 prostate cancer cells.

Evidence suggests that 17beta-estradiol (E2) contributes to the risk of prostate cancer (PCa), whereas the phytochemicals genistein from soy and 3,3'-diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, decrease the risk of PCa. This study examined the potential of these phytochemicals to reduce the adverse effects of E2 on PCa. In LNCaP PCa cells (E2 sensitive), DIM decreased E2-induced proliferation. Genistein increased proliferation at low concentrations and decreased proliferation at higher concentrations; DIM abolished the increased proliferation by genistein. The E2 stimulation in LNCaP cells was consistent with dependence on the androgen receptor, as evidenced by the inhibition of E2-induced proliferation with the antiandrogen casodex, E2 stimulation of an androgen response element luciferase reporter, and E2 stimulation of prostate-specific antigen (PSA) protein expression. Both genistein and DIM abrogated the E2 stimulation of PSA. Genistein and DIM altered major E2 metabolism pathways in LNCaP and PC-3 (E2 insensitive) PCa cells by increasing the expression of the 2-hydoxylation enzyme cytochrome P450 1A1 (CYP1A1) and the O-methylating enzyme catechol-o-methyltransferase (COMT) as determined by real-time RT-PCR. The increase in COMT mRNA occurred only when the combination of DIM and genistein (15 micromol/L) was used. Quantitation by MS indicated increased 2-hydroxyestrogen and decreased 16alpha-hydroxyestrone, a result that should result in less estrogenicity and increased amounts of the anticancer metabolite 2-methoxyestrone. We conclude that DIM and genistein decrease the effects of E2 that have the potential to promote PCa.

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11.

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.

Variants in estrogen metabolism and biosynthesis genes and urinary estrogen metabolites in women with a family history of breast cancer.

We examined associations between polymorphisms in genes related to estrogen metabolism (CYP1B1 codon 432G --> C rs#1056836, CYP1B1 codon 453A --> G rs#1800440, COMT codon 158G --> A rs#4680) and biosynthesis (CYP17 T --> C promoter rs#743572, CYP19 exon 4 TTTA repeat) and urinary estrogen metabolites (2-hydroxyestrogens (2-OHE), 16alpha-hydroxyestrone (16alpha-OHE1), and their ratio) in a pilot study of 64 pre- and post-menopausal women with a family history of breast cancer. Women were participants in the Metropolitan New York Registry of Breast Cancer Families, one of six international sites of the National Cancer Institute's Breast Cancer Family Registry. We used linear regression to examine the effects of genetic variants on log-transformed urinary estrogen metabolites. After adjusting for menopausal status, BMI, and age, carriers of the CYP1B1 codon 453G variant allele had 31.0% lower levels of 2-OHE (P-value = 0.05) and 40.2% lower levels of 16alpha-OHE1 (P = 0.01). Results were similar after restricting the analyses to pre-menopausal women (n = 41). Consistent with other studies, among pre-menopausal women, carriers of the COMT codon 158A variant allele had increased 2-OHE levels (P = 0.03) and an increased 2-OHE/16alpha-OHE1 ratio (P = 0.04); carriers of the CYP17 C promoter variant allele had increased 2-OHE levels (P = 0.08). To our knowledge this is the first report showing associations between the CYP1B1 codon 453G variant allele and urinary 2-OHE and 16alpha-OHE1 metabolites. Further larger studies should be conducted to confirm these results. Future identification of individuals with genetic polymorphisms that affect estrogen metabolism and biosynthesis may help characterize women at higher breast cancer risk and could guide breast cancer prevention strategies for those individuals.

A hormonal association between estrogen metabolism and proliferative thyroid disease.

OBJECTIVE: To illustrate a relationship between proliferative thyroid disease and estrogen metabolism through the analysis of urinary estrogen metabolites. STUDY DESIGN AND SETTING: Case-control study of 49 subjects with proliferative thyroid disorders and matching them to 49 controls. Urinary estrogen metabolite ratios were obtained, measuring 2-hydroxyestrone, an anti-proliferative metabolite, to 16alpha-hydroxyestrone, a proliferative metabolite. The patients were stratified into low (0 to 1.00), medium (1.01 to 2.00), or high (>2.00) groups according to their estrogen metabolite ratio. RESULTS: Fifty-one percent (25 of 49) of the cases had a low 2/16 ratio compared to 31% (15 of 49) in the control group while 20% (10 of 49) of the control group had a high 2/16 ratio as compared to 8% (4 of 49) in the case group (P value < 0.05). CONCLUSIONS: Increased 16alpha-hydroxyestrone activity compared to 2-hydroxyestrone activity appears to be associated with proliferative thyroid disease. SIGNIFICANCE: Further study of estrogen metabolites in relation to proliferative thyroid disease is warranted and may lead to implications for new treatment modalities for proliferative thyroid disease. EBM RATING: B-3b.