Tissue invasion and metastasis: Molecular, biological and clinical perspectives.

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), ß-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-ß), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.

Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer.

In response to cell stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). Little was known about the potential role in cancer of a different mode of UPR activation, anticipatory activation of the UPR prior to accumulation of unfolded protein or cell stress. We show that estrogen, acting via estrogen receptor α (ERα), induces rapid anticipatory activation of the UPR, resulting in increased production of the antiapoptotic chaperone BiP/GRP78, preparing cancer cells for the increased protein production required for subsequent estrogen-ERα-induced cell proliferation. In ERα-containing cancer cells, the estrogen, 17ß-estradiol (E2) activates the UPR through a phospholipase C γ (PLCγ)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ERα blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLCγ, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E2-ERα activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6α, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E2-ERα-stimulated cell proliferation. Mild and transient UPR activation by estrogen promotes an adaptive UPR response that protects cells against subsequent UPR-mediated apoptosis. Analysis of data from ERα(+) breast cancers demonstrates elevated expression of a UPR gene signature that is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen therapy, reduced time to recurrence and poor survival. Thus, as an early component of the E2-ERα proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for estrogens in cancer cell proliferation and resistance to therapy.

Association between absolute tumor burden and serum bone-specific alkaline phosphatase in canine appendicular osteosarcoma.

BACKGROUND: In dogs with appendicular osteosarcoma (OSA), increased pretreatment serum bone-specific alkaline phosphatase (BALP) activity is a negative prognostic factor, associated with shorter disease-free intervals and survival times, but a biologic basis for observed differential serum BALP activities in canine OSA patients remains incompletely defined. OBJECTIVE: Serum BALP activity will correlate with absolute tumor burden in dogs with OSA. ANIMALS: This study included 96 client-owned dogs with appendicular OSA. METHODS: In canine OSA cell lines, the expression and membranous release of BALP was evaluated in vitro. The correlation between serum BALP activity and radiographic primary tumor size was evaluated in OSA-bearing dogs. In dogs developing visceral OSA metastases, serial changes in serum BALP activities were evaluated in relation to progression of macroscopic metastases, and visceral metastatic OSA cells were evaluated for BALP expression. RESULTS: In vitro, BALP expression was not associated with either tumorigenic or metastatic phenotype, rather the quantity of membranous BALP released was proportional with cell density. In dogs devoid of macroscopic metastases, there was a positive correlation between serum BALP activity and absolute primary tumor size. In dogs with progressive OSA metastases, serum BALP activity increased and coincided with the development of macroscopic metastases. OSA cells derived from visceral metastatic lesions retained BALP expression. CONCLUSIONS AND CLINICAL IMPORTANCE: Tumor burden is a determinant of serum BALP activity in dogs with appendicular OSA. The association between increased pretreatment BALP activity and negative clinical prognosis may simply be attributed to greater initial tumor burden, and consequently more advanced tumor stage.

Body mass influences cortical bone mass independent of leptin signaling.

Obesity in humans is associated with increased bone mass. Leptin, a hormone produced by fat cells, functions as a sentinel of energy balance, and may mediate the putative positive effects of body mass on bone. We performed studies in male C57Bl/6 wild type (WT) and leptin-deficient ob/ob mice to determine whether body mass gain induced by high fat intake increases bone mass and, if so, whether this requires central leptin signaling. The relationship between body mass and bone mass and architecture was evaluated in 9-week-old and 24-week-old WT mice fed a regular mouse diet. Femora and lumbar vertebrae were analyzed by micro computed tomography. In subsequent studies, slowly and rapidly growing ob/ob mice were injected in the hypothalamus with a recombinant adeno-associated virus containing the leptin gene (rAAV-lep) or a control vector, rAAV-GFP (green fluorescent protein). The mice were maintained on a regular control diet for 5 or 7 weeks and then subdivided into groups and either continued on the control diet or fed a high fat diet (45% of kcal from fat) for 8 weeks. In the WT mice, femoral and vertebral bone mass was positively correlated with body mass (Pearson's r=0.65-0.88 depending on endpoint). rAAV-lep therapy dramatically decreased body mass (-61%) but increased femur length. However, in the distal femur and lumbar vertebra, rAAV-lep therapy reduced cancellous bone volume/tissue volume, trabecular number and trabecular thickness, and increased trabecular spacing. The high fat diet increased body mass, irrespective of vector treatment. Total femur bone volume, length, cross-sectional volume, and cortical volume and thickness were increased in mice with increased body mass, independent of rAAV treatment. In the distal femur, increased body mass had no effect on cancellous architecture and there were no vector x body mass interactions. In WT mice, increased body mass resulted in increased (+33%) vertebral cancellous bone volume/tissue volume. Increased body mass had minimal independent effect on cancellous vertebral bone mass in ob/ob mice. Taken together, these findings suggest that increased body mass has a positive effect on femur cortical bone mass that is independent of leptin signaling.

Phytoestrogens and breast cancer: a complex story.

Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products as a constituent of complex mixtures of bioactive compounds, whose matrix profiles play an important role in determining the overall oestrogenic bioactivity of genistein. We review data on how the profile of soy bioactive compounds can modulate genistein-stimulated oestrogen-dependent tumour growth. Our research has focused on the effects of dietary genistein on the growth of oestrogen (E)-dependent mammary tumours both in vitro and in vivo. Genistein enhances the proliferation of E-dependent human breast cancer tumour growth. In a similar manner, dietary genistein stimulates tumour growth in the chemically-induced (NMU) mammary cancer rodent model. Genistin, the glycoside of genistein, simulates growth similar to that of genistein and withdrawal of either genistein or genistin results in tumour regression. The extent of soy processing modulates the effects of dietary genistein in vivo as soy protein isolate, a highly purified and widely used source of protein that is processed to contain low, medium, and high amounts of isoflavones, stimulate the growth of the E-dependent mammary tumours in a dose dependent manner. In contrast to the more purified diets, studies with soy flour of equivalent genistein levels did not stimulate the growth of E-dependent breast cancer tumours in vivo. However, the size of these tumours also did not regress as is observed in control groups in which oestrogen and genistein have been withdrawn. The expression of the oestrogen-target genes of pS2, progesterone receptor, and cyclin D1 correlates with the growth of E-dependent tumours and has been consistently observed to be induced in response to treatment with dietary genistein. To evaluate whether dietary genistein interacts with current anti-oestrogen breast cancer therapies such as tamoxifen (TAM), we implanted E-dependent tumours into ovariectomized athymic mice and administered oestradiol, oestradiol plus TAM, or oestradiol, TAM, and dietary genistein. In these studies dietary genistein was able to negate the inhibitory effect of TAM on E-stimulated tumour growth. In summary, genistein can act as an oestrogen agonist resulting in proliferation of E-dependent human breast cancer tumours in vivo and its activity can be modulated by the presence of other bioactive components in complex soy foods. Additionally, dietary genistein can negate the inhibitory effects of TAM on E-stimulated growth of MCF-7 cell tumours implanted into ovariectomized athymic mice.

The phytoestrogen genistein suppresses cell-mediated immunity in mice.

The soy phytoestrogen, genistein, induces thymic atrophy when administered to ovariectomized mice by injection or in the diet. Injected genistein also causes decreased humoral immunity, but the effects of genistein on cell-mediated immunity have not been addressed. Here we examined effects of injected and dietary genistein on cell-mediated immune responses. Female C57BL/6 mice (25- to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily subcutaneous injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/kg genistein plus the anti-estrogen ICI 182,780 (5 mg/kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0.05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4(+) and CD8(+) T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cell-mediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. Further work is needed to determine if dietary genistein and phytoestrogen exposure can produce effects on cell-mediated immunity in humans or other animals under various nutritional conditions.

Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice.

Previously our laboratory has shown that the soy isoflavone, genistein, stimulates growth of human breast cancer (MCF-7) cells in vivo and in vitro. In this study, the dose-response analysis of genistein at the physiologically achievable concentration range between 125 and 1,000 microg/g in the diet was conducted in ovariectomized athymic nude mice implanted with MCF-7 cells. We hypothesized that genistein at this concentration range can stimulate dose-dependently the breast tumor growth, cell proliferation and an estrogen-responsive pS2 gene induction. Tumor size and body weight were monitored weekly. At completion of the study, we analyzed cellular proliferation of tumors using incorporation of BrdU, pS2 expression of tumors using a Northern blot analysis and total genistein level in plasma using liquid chromatography-isotope dilution mass spectrometry (LC-ES/MS). Dietary genistein (> or = 250 microg/g) increased tumor size in a dose-dependent manner [8.4x the negative control (NC) group in the 250 microg/g group, 12.0x in the 500 microg/g group, 20.2x in the 1,000 microg/g group and 23.2x in the positive control (PC) group]. The percentage of proliferating cells was significantly increased by genistein at and above 250 microg/g (5.3x the NC group in the 250 microg/g, 5.6x in the 500 microg/g, 5.0x in the 1,000 microg/g and 4.8x in the PC group). Expression of pS2 mRNA was also significantly increased with increasing dietary genistein levels (11.25x the NC group in the 500 microg/g group and 15.84x in the 1,000 microg/g group). Total plasma genistein concentrations were between 0.39 and 3.36 micromol/L in mice fed between 125 and 1,000 microg/g genistein. In conclusion, dietary treatment with genistein at physiological concentrations produces blood levels of genistein sufficient to stimulate estrogenic effects, such as breast tumor growth, cellular proliferation and pS2 expression in athymic mice in a dose-responsive manner similar to that seen in vitro.

Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein.

The estrogenic soy isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo. Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogen-dependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.

Soy diets containing varying amounts of genistein stimulate growth of estrogen-dependent (MCF-7) tumors in a dose-dependent manner.

We have demonstrated that the isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo (C. Y. Hsieh et al., Cancer Res., 58: 3833-3838, 1998). The isoflavones are a group of phytoestrogens that are present in high concentrations in soy. Whether consumption of genistein from soy protein will have similar effects on estrogen-dependent tumor growth as pure genistein has not been investigated in the athymic mouse tumor implant model. Depending on processing, soy protein isolates vary widely in concentrations of genistein. We hypothesize that soy isolates containing different concentrations of genistein will stimulate the growth of estrogen-dependent cells in vivo in a dose-dependent manner. To test this hypothesis we conducted experiments in which these soy protein isolates were fed to athymic mice implanted s.c. with estrogen-dependent tumors. Genistein content (aglycone equivalent) of the soy isolate diets were 15, 150, or 300 ppm. Positive (with 17beta-estradiol pellet implant) and negative (no 17beta-estradiol) control groups received casein-based (isoflavone-free) diets. Tumor size was measured weekly. At completion of the study animals were killed and tumors collected for evaluation of cellular proliferation and estrogen-dependent gene expression. Incorporation of bromodeoxyuridine into cellular DNA was used as an indicator of cell proliferation, and pS2 mRNA was used as an estrogen-responsive gene. Soy protein diets containing varying amounts of genistein increased estrogen-dependent tumor growth in a dose-dependent manner. Cell proliferation was greatest in tumors of animals given estrogen or dietary genistein (150 and 300 ppm). Expression of pS2 was increased in tumors from animals consuming dietary genistein (150 and 300 ppm). Here we present new information that soy protein isolates containing increasing concentrations of genistein stimulate the growth of estrogen-dependent breast cancer cells in vivo in a dose-dependent manner.

Estrogenic effects of extracts from cabbage, fermented cabbage, and acidified brussels sprouts on growth and gene expression of estrogen-dependent human breast cancer (MCF-7) cells.

Cruciferous vegetable extracts from freeze-dried cabbage (FDC), freeze-dried fermented cabbage (FDS), and acidified Brussels sprouts (ABS) were prepared by exhaustive extraction with ethyl acetate. Estrogenic and antiestrogenic effects of these extracts were analyzed. To identify whether the extracts are potential estrogen receptor (ER) ligands that can act as agonists or antagonists, the binding affinity of extracts for the ER was measured using a competitive radiometric binding assay. The extracts bound with low affinity to the ER, and the relative binding affinity is estradiol > FDS > FDC > ABS. These extracts were evaluated for their estrogenic and antiestrogenic activities in estrogen-dependent human breast cancer (MCF-7) cells using as endpoints proliferation and induction of estrogen-responsive pS2 gene expression, which was analyzed using Northern blot assay. At low concentrations (5-25 ng/mL) all of the extracts reduced 1 nM estradiol-induced MCF-7 cell proliferation. Extracts at 25 ng/mL also inhibited estradiol-induced pS2 mRNA expression. At higher extract concentrations (50 ng/mL-25 microg/mL), however, increased proliferation in MCF-7 cells was observed. Similarly, expression of the pS2 gene was induced by higher extract concentrations (0.25-25 microg/mL). The pure estrogen antagonist, ICI 182,780, suppressed the cell proliferation induced by the extracts as well as by estradiol and also the induction of pS2 expression by the extracts. The ER subtype-selective activities of FDC and FDS were analyzed using a transfection assay in human endometrial adenocarcinoma (HEC-1) cells. FDS acted as an ERalpha-selective agonist while FDC fully activated both ER-alpha and ER-beta. Growth of the ER-negative MDA-231 cells was not affected by the extracts or by estradiol. This study demonstrates that cruciferous vegetable extracts act bifunctionally, like an antiestrogen at low concentrations and an estrogen agonist at high concentrations.

Genistein inhibits growth of estrogen-independent human breast cancer cells in culture but not in athymic mice.

The studies presented were conducted to assess the effect of the soy isoflavone genistein on proliferation of estrogen-independent human breast cancer cells (MDA-MB-231) in vitro and in vivo. Genistein (20 mcmol/L) inhibited cell proliferation in vitro by approximately 50%. Cell cycle progression was blocked in G(2)/M with 40 and 80 mcmol/L genistein. To evaluate the effect of dietary genistein on tumor growth in vivo, genistein was fed to female athymic mice inoculated with MDA-MB-231 cells. After solid tumor masses had formed, mice were fed genistein at a dose (750 mcg/g AIN-93G diet), shown to produce a total plasma genistein concentration of approximately 1 mcmol/L. This dose of genistein did not significantly (P > 0.05) alter tumor growth. Studies were then conducted to assess the effect of dietary genistein on initial tumor development and growth. Genistein (750 mcg/g AIN-93G diet), fed 3 d before cells were inoculated into mice, did not significantly (P > 0.05) inhibit tumor formation or growth. The plasma concentration of genistein in mice fed this dose of dietary genistein (750 mcg/g AIN-93G diet) does not appear sufficient to inhibit tumor formation or growth. Dietary genistein at 750 mcg/g AIN-93G diet does not inhibit tumor formation or growth. Additional studies were conducted to determine the effect of dietary dosages ranging from 0 to 6000 mcg/g AIN-93G diet on plasma genistein concentration. Plasma genistein concentration increased in a dose-dependent manner up to 7 mcmol/L at 6000 mcg/g AIN-93G diet. These data suggest that although genistein inhibits cancer cell growth in vitro, it is unlikely that the plasma concentration required to inhibit cancer cell growth in vivo can be achieved from a dietary dosage of genistein.

Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo.

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.

Multigenerational study of the effects of consumption of PCB-contaminated carp from Saginaw Bay, Lake Huron, on mink. 1. Effects on mink reproduction, kit growth and survival, and selected biological parameters.

This study was conducted to determine the multigenerational effects of consumption of PCB-contaminated carp (Cyprinus carpio) from Saginaw Bay (Lake Huron) on mink (Mustela vison) reproduction and health and to examine selected biomarkers as potential indicators of polyhalogenated hydrocarbon toxicity in mink. The mink were fed diets formulated to provide 0 (control), 0.25, 0.5, or 1.0 ppm polychlorinated biphenyls (PCBs) through substitution of Saginaw Bay carp for ocean fish in the diets. To determine whether the effects of PCB exposure were permanent, half of the parental (P1) animals were switched from their respective treatment diets to the control diet after whelping the first of two F1 generations. Effects of in utero and lactational exposure to PCBs on subsequent reproductive performance of the F1 animals were examined by switching half of the first-year F1 offspring (kits) to the control diet at weaning, while the other half was continued on their parental diet (continuous exposure). Continuous exposure to 0.25 ppm, or more, of PCBs delayed the onset of estrus (as determined by vulvar swelling and time of mating) and lessened the whelping rate. Litters whelped by females continually exposed to 0.5 ppm, or more, of PCBs had greater mortality and lesser body weights than controls. Continuous exposure to 1.0 ppm PCBs had a variable effect on serum T4 and T3 concentrations. Compared to the controls, there were significant differences in kidney, liver, brain, spleen, heart, and thyroid gland weights of the mink continually exposed to 1.0 ppm PCBs. There was an increase in the incidence of periportal and diffuse vacuolar hepatocellular lipidosis in the P1 mink with continuous exposure to increasing concentrations of PCBs. Plasma and liver PCB concentrations of the adult and kit mink were, in general, directly related to the dietary concentration of PCBs and the duration and time of exposure. Short-term parental exposure to PCBs had detrimental effects on survival of subsequent generations of mink conceived months after the parents were placed on "clean" feed. The lowest observed adverse effect level (LOAEL) for dietary PCBs in this study was 0.25 ppm.

Multigenerational study of the effects of consumption of PCB-contaminated carp from Saginaw Bay, Lake Huron, on mink. 2. Liver PCB concentration and induction of hepatic cytochrome P-450 activity as a potential biomarker for PCB exposure.

This study examined the effect of polychlorinated biphenyls (PCBs) from Saginaw Bay (Lake Huron) carp on the hepatic cytochrome P-450 activity in mink (Mustela vison). Hepatic cytochrome P-450 activities are of interest for their possible use as biomarkers to indicate consumption and biological effects of PCBs in the environment. Adult mink were fed diets containing ocean fish (control diet, 0.0 ppm) or Saginaw Bay carp toprovide 0.25, 0.5, or 1.0 ppm PCBs. Mink were bred after 3 mo of exposure, and half of the parental mink (P1) and kits (F1-1) previously consuming diets containing Saginaw Bay carp were switched to control diet at weaning of the F1-1 kits. P1 and F1-1 mink were then bred within their age and dietary groups after 15 mo of exposure, to produce the second-year F1 (F1-2) and F2 kits. Mink were killed when the new kits were weaned. Transfer of half the animals to the control diet examined whether the effects of the PCB-containing diet on hepatic cytochrome P-450 activity were permanent. Continual exposure to diets containing PCBs from Saginaw Bay carp induced cytochrome P-450 activity in a generally dose-dependent manner. Cytochrome P-450 activity was not different from untreated controls in animals switched to the control diet from the PCB-containing diet. The response of cytochrome P-4501A1 (EROD) activity in a dose-dependent manner and the lack of induction after transfer to noncontaminated diets suggest that this hepatic enzyme activity is a potential biomarker for current exposure to PCBs and other similar cytochrome P-450 inducers.

Multigenerational study of the effects of consumption of PCB-contaminated carp from Saginaw Bay, Lake Huron, on mink. 3. Estrogen receptor and progesterone receptor concentrations, and potential correlation with dietary PCB consumption.

Mink (Mustela vison) were fed diets containing ocean fish (control diet, 0.0 ppm polychlorinated biphenyls, PCBs) or Saginaw Bay carp to provide 0.25, 0.5, or 1.0 ppm PCBs to examine the effect of PCBs on homeostasis of binding sites for ovarian steroid hormones. Ranch-raised mink fed Great Lakes fish contaminated with PCBs, or treated with PCBs directly, have demonstrated reproductive impairment including anovulation, fetal resorption, delayed ovulation, increased gestation, and decreased litter size. Previous studies have demonstrated that estrogen and progesterone levels are unaltered in mink treated with PCBs, suggesting that the effect of PCBs on reproduction is not mediated through alterations in hormone homeostasis. In vitro studies have demonstrated that the most likely means by which PCBs exert antiestrogenic ability is through a down-regulation of the estrogen receptor in normally estrogen-responsive tissues such as liver and uterus. Hepatic and uterine estrogen binding site concentrations were measured in female mink consuming diets containing PCBs for up to 18 mo at up to 1 ppm. Hepatic estrogen binding site concentrations generally decreased with increasing dietary PCB concentrations. Uterine estrogen binding site concentration did not decrease in these animals. Uterine progesterone receptor concentration also did not change with increasing PCB consumption. In total, the response of hepatic and uterine estrogen and uterine progesterone binding sites in mink fed diets containing Saginaw Bay carp suggests that concentrations of PCBs available to uterine tissue may not have been sufficient to decrease uterine estrogen receptor, despite their effect on hepatic estrogen receptor.

Hydroxylated polychlorinated biphenyl metabolites are anti-estrogenic in a stably transfected human breast adenocarcinoma (MCF7) cell line.

Hydroxylated metabolites of polychlorinated biphenyls (OHCBs) have been identified in blood of marine mammals, fish-eating birds, and humans at concentrations in some cases exceeding those of the unmetabolized polychlorinated biphenyls (PCBs). OHCBs have been associated with inhibition of vitamin A and thyroxin transport, estrogenicity in a mouse uterotrophic assay, and feminization of male turtle sexual development. OHCBs, representing both environmentally derived and laboratory exposure-derived metabolites, were tested in an in vitro bioassay utilizing an estrogen-responsive human breast adenocarcinoma cell line (MCF7-LUC) stably transfected with a luciferase reporter gene linked to estrogen responsive elements. OHCB activity was tested at three different media concentrations of 17beta-estradiol (E2), comparing the concentration-response curves using charcoal-stripped medium (0.0009 nM E2), and two physiologically relevant E2 concentrations (0.1 and 1.0 nM E2). Eleven of 13 OHCBs tested were anti-estrogenic. Evidence for an estrogen receptor mediated mechanism of action was apparent for only two OHCBs-4-OH-2',3,3',4',5,5'-Cl6-biphenyl and 4,4'-(OH)2-3,3',5,5'-Cl4-biphenyl. These two have not been identified in environmental samples. The remaining OHCBs exhibited "anti-estrogenicity" that was related to their effect on cell viability and, therefore, cannot be described as exhibiting "hormone disruption" solely by an estrogen receptor mediated mechanism. OHCB anti-estrogenic activity was eliminated in the presence of E2 concentrations normally found in humans, except for 4,4'(OH)2-3,3',5,5'-Cl4-biphenyl. 4-OH-2',3',4',5'-Cl4-biphenyl and 4-OH-2',4',6'-Cl3-biphenyl were partial estrogen agonists, exhibiting weak estrogenicity in the presence of 0.0009 nM E2 and weak anti-estrogenicity in the presence of 0.1 and 1 nM E2. Human metabolites of PCBs were not estrogenic in MCF7 cells.

A unique myosin isoform transition in cat striated external anal sphincter muscle induced by denervation.

Denervation of limb and trunk muscles leads to characteristic changes in their biochemical properties. However, the effects of denervation of the striated external anal sphincter (EAS) muscle have not been studied, even though denervation is a common etiology for fecal incontinence. The present study reports effects ofdenervation of the cat EAS and compares them with changes in a denervated limb muscle [cat extensor digitorum longus (EDL)]. Nerves supplying the EAS and the EDL were sectioned in anesthetized cats, which were allowed to recover and live for 10, 30, or 60 days. Their muscles were then excised, and myofibrillar proteins were isolated. Myosin heavy chain (MHC) isoform composition was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDL and EAS showed progressive increases in MHC type IIA and progressive decreases in MHC type IIB at 10, 30, and 60 days. MHC type I progressively increased in the EDL but showed no change in the EAS. We hypothesize that this distinctive transition is related to the unique embryological origin and function of the EAS.

Dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the hypothalamic/pituitary axis in rats.

These studies were undertaken to assess the estrogenic and antiestrogenic effects of dietary genistein. To determine estrogenic effects, genistein was mixed into a modified AIN-76 or AIN-93G semipurified diet at 0 (negative control), 150, 375 or 750 microg/g and 17, beta-estradiol at 1.0 microg/g and fed to ovariectomized 70-d-old Sprague-Dawley rats. Estrogenic potency was determined by analyzing uterine weight, mammary gland development, plasma prolactin and expression of uterine c-fos. Dietary genistein (375 and 750 microg/g) increased uterine wet and dry weights (P < 0.05). Mammary gland regression following ovariectomy was significantly inhibited by dietary genistein at 750 microg/g (P < 0.05). Plasma prolactin was significantly greater in ovariectomized rats fed genistein (750 microg/g) compared with comparable rats not receiving genistein. The relative binding affinity of genistein to the estrogen receptor (ER) was 0.01 that of estradiol. Genistein (750 microg/g) induced the uterine expression of c-fos. To evaluate potential antiestrogenic effects, genistein and estradiol were mixed into the modified AIN diets at the doses noted above and fed to ovariectomized rats. Dietary genistein (375 or 750 microg/g) did not inhibit the effects of estradiol on uterine weight, mammary gland development or plasma prolactin. Serum concentration of total genistein (conjugated plus free) in rats fed 750 microg/g was 2.2 micromol/L and free genistein was 0.4 micromol/L. Administration of dietary genistein at 750 microg/g can exert estrogenic effects in the uterus, mammary gland and hypothalamic/pituitary axis. Dietary genistein (750 microg/g) did not antagonize the action of estradiol in estradiol-supplemented ovariectomized rats or in intact rats.

Effects of zearalenone and/or tamoxifen on swine and mink reproduction.

Tamoxifen (TAM), which binds to estrogen receptors and can act as an estrogen antagonist, was incorporated into the diets of swine and mink to determine if it would ameliorate the effects of the estrogenic mycotoxin zearalenone (ZEN). Sows and female mink were fed diets containing 2 ppm (swine) or 20 ppm (mink) ZEN and/or 10 ppm TAM from day 30 of gestation through weaning (swine) or from 2 months prior to breeding through weaning (mink). The diets containing ZEN and/or TAM did not adversely affect reproduction in the sows. Although some hyperestrogenic effects on testes, uterine and ovarian weights were observed in the F1 piglets at 21 days of age, subsequent breeding performance was not affected. All the female mink exposed to ZEN mated, but only 25% whelped. No mink fed TAM (singly or in combination with ZEN) mated. Necropsy of these unmated females fed TAM revealed consistent severe pyometra. Histological examination of the reproductive tracts of the ZEN, TAM and ZEN + TAM-treated mink showed similar alterations, including ovarian follicular atrophy and degeneration, and mild to severe uterine atrophy, pyometra and endometritis. The results of these studies indicate that TAM was not effective in ameliorating the hyperestrogenic effects of ZEN in swine and mink, but rather it acted as an estrogen agonist.

Elevated neuronal c-Fos-like immunoreactivity and messenger ribonucleic acid (mRNA) in genetically obese (ob/ob) mice.

Adult genetically obese (ob/ob) mice display a number of metabolic alterations, the primary cause of which may be a defect in their central nervous system (CNS). The protein encoded by the protooncogene c-fos, c-Fos, functions as a nuclear transcription factor, and also serves as a marker of neuronal activity. The specific objectives of this study were (1) to use c-Fos immunohistochemistry to identify regions with altered neuronal activity in 6-7 week old male lean and ob/ob mice; (2) to examine c-fos relative mRNA abundance by northern blot analysis in brains of these mice and compare it with that of neuropeptide Y (NPY), a peptide well known to alter feeding and (3) determine changes in c-Fos immunoreactivity and mRNA caused by food deprivation. Fos-like immunoreactivity (FLI) tended to be higher in ad libitum fed ob/ob mice than in lean controls in most brain regions examined. The most prominent and consistent differences were in the paraventricular nuclei (PVN) where the numbers of Fos-positive nuclei were approximately 3 fold higher in ob/ob mice. Food deprivation for 24 h increased FLI in the PVN in lean mice but did not further augment FLI in the PVN of ob/ob mice. Arcuate nuclei of lean and ob/ob mice showed minimal FLI staining under ad libitum fed conditions. Food deprivation however, induced FLI in arcuate nuclei of both lean and ob/ob mice. The abundance of c-fos mRNA in whole brain of ob/ob mice averaged several fold higher than in leans under both fed and fasted conditions.(ABSTRACT TRUNCATED AT 250 WORDS)