Ruth S. MacDonald

Neuroprotective mechanisms of curcumin against cerebral ischemia-induced neuronal apoptosis and behavioral deficits.

Increased oxidative stress has been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In recent years, there has been increasing interest in investigating polyphenols from botanical source for possible neuroprotective effects against neurodegenerative diseases. In this study, we investigated the mechanisms underlying the neuroprotective effects of curcumin, a potent polyphenol antioxidant enriched in tumeric. Global cerebral ischemia was induced in Mongolian gerbils by transient occlusion of the common carotid arteries. Histochemical analysis indicated extensive neuronal death together with increased reactive astrocytes and microglial cells in the hippocampal CA1 area at 4 days after I/R. These ischemic changes were preceded by a rapid increase in lipid peroxidation and followed by decrease in mitochondrial membrane potential, increased cytochrome c release, and subsequently caspase‐3 activation and apoptosis. Administration of curcumin by i.p. injections (30 mg/kg body wt) or by supplementation to the AIN76 diet (2.0 g/kg diet) for 2 months significantly attenuated ischemia‐induced neuronal death as well as glial activation. Curcumin administration also decreased lipid peroxidation, mitochondrial dysfunction, and the apoptotic indices. The biochemical changes resulting from curcumin also correlated well with its ability to ameliorate the changes in locomotor activity induced by I/R. Bioavailability study indicated a rapid increase in curcumin in plasma and brain within 1 hr after treatment. Together, these findings attribute the neuroprotective effect of curcumin against I/R‐induced neuronal damage to its antioxidant capacity in reducing oxidative stress and the signaling cascade leading to apoptotic cell death.

Phytoestrogens in Common Herbs Regulate Prostate Cancer Cell Growth in Vitro

Abstract: Prostate cancer is an important public health problem in the United States. Seven phytoestrogens found in common herbal products were screened for estrogen receptor binding and growth inhibition of androgen-insensitive (PC-3) and androgen-sensitive (LNCaP) human prostate tumor cells. In a competitive 3H-estradiol ligand binding assay using mouse uterine cytosol, 2.5 μM quercetin, baicalein, genistein, epigallocatechin gallate (EGCG), and curcumin displaced > 85% of estradiol binding, whereas apigenin and resveratrol displaced > 40%. From growth inhibition studies in LNCaP cells, apigenin and curcumin were the most potent inhibitors of cell growth, and EGCG and baicalein were the least potent. In PC-3 cells, curcumin was the most potent inhibitor of cell growth, and EGCG was the least potent. In both cell lines, significant arrest of the cell cycle in S phase was induced by resveratrol and EGCG and in G2M phase by quercetin, baicalein, apigenin, genistein, and curcumin. Induction of apoptosis was induced by all of the 7 compounds in the 2 cell lines as shown by TUNEL and DNA fragmentation assays. Androgen responsiveness of the cell lines did not correlate with cellular response to the phytoestrogens. In conclusion, these 7 phytoestrogens, through different mechanisms, are effective inhibitors of prostate tumor cell growth.

Soy Isoflavones Have an Antiestrogenic Effect and Alter Mammary Promoter Hypermethylation in Healthy Premenopausal Women

We determined if soy isoflavones have dose-related estrogenic and methylation effects. Thirty-four healthy premenopausal women were randomized to 40 mg or 140 mg isoflavones daily through one menstrual cycle. Breast specific and systemic estrogenic effects were assessed measuring the estrogenic marker complement (C)3 and changes in cytology, whereas methylation assessment of 5 cancer related genes (p16, RASSF1A, RAR β 2, ER, and CCND2) was performed on intraductal specimens. Serum genistein significantly increased after consuming both isoflavone doses. Cytology did not significantly change at either isoflavone dose. Serum C3 levels posttreatment were inversely related to change in serum genistein ( r =–0.76, P = 0.0045) in women consuming low but not high dose isoflavones. The RAR β 2 hypermethylation increase posttreatment correlated with the posttreatment genistein level considering the entire group ( r = 0.67, P = 0.0017) and those receiving high-dose isoflavones ( r = 0.68, P = 0.021). At the low but not the high isoflavone dose, CCND2 hypermethylation increase correlated with posttreatment genistein levels ( r = 0.79, P = 0.011). In summary, the inverse correlation between C3 and genistein suggests an antiestrogenic effect. Isoflavones induced dose-specific changes in RAR β 2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.

Dietary Genistein Increased DMBA-Induced Mammary Adenocarcinoma in Wild-Type, but Not ERαKO, Mice

Dietary supplements containing concentrates of plant-derived estrogens are being increasingly used by consumers as alternatives for hormone replacement therapy, for treatment of menopausal symptoms, and as cancer preventives. The effect of dietary genistein on dimethylbenz[a]anthracene (DMBA)-induced mammary tumor development was investigated in wild-type (ERαWT) and estrogen receptor-α knockout (ERαKO) mice. ERαWT and ERαKO mice were fed a casein-based diet containing 0 or 1 g genistein/kg diet from weaning. Tumors were induced by oral administration of DMBA and subscapular implantation of medroxyprogesterone acetate. No tumors were observed in ERαKO mice. In ERαWT mice, dietary intake of genistein influenced tumor development, enhancing anaplasia of mammary cancer. Mice consuming genistein expressed malignant mammary adenocarcinoma, whereas benign adenomas were observed in mice fed the control diet. Dietary intake was also influenced by genistein, with ERαWT and ERαKO mice fed genistein consuming less food (p < 0.0001) and subsequently weighing less than mice fed the control diet (p < 0.0001). Significant differences in food intake by genotype were also observed (p = 0.0017), with ERαKO mice consuming less than ERαWT mice. Overall, this study found no protective effect of genistein on DMBA-induced mammary tumors in mice and suggests a potential adverse effect on tumor development when high levels of genistein are consumed.

Effects of Diets Enriched in Omega-3 and Omega-6 Polyunsaturated Fatty Acids on Offspring Sex-Ratio and Maternal Behavior in Mice

Abstract There have been many trials describing the effects of polyunsaturated fatty acids (PUFA) on fecundity, neonatal development, and maternal behavior in humans, but few controlled studies in rodents. We examined the effects of a maternal diet high in omega 3 (N-3) or omega 6 (N-6) PUFA on NIH Swiss mice. Female mice were ad libitum fed one of three complete and balanced diets (N-3, enriched in menhaden oil; N-6, enriched in corn oil; C, control diet, Purina 5015) from age 4 wk until the end of the study. Mice were bred at ∼19 wk and 27 wk of age, providing a total of 838 pups from 129 litters in two experiments. After weaning their pups from parity 1, behavior of dams was assessed on elevated-plus and open-field mazes. Although the fraction of male pups from the N-3 and C groups was not different from 0.5, dams on the N-6 diet birthed more daughters than sons (213 vs. 133; P < 0.001). Although maternal stress has been reported to favor birth of daughters, the behavior of N-6 dams was not different from controls. By contrast, the N-3 dams displayed greater anxiety, spending less time in the open arms and more time in the closed arms of the elevated maze and traveling less distance and exhibiting less exploratory behavior in the open field (P < 0.05). N-3 dams tended to produce smaller litters than C dams, and N-3-suckled pups gained less weight (P < 0.05). In conclusion, the N-3 diet had negative effects on murine fecundity and maternal behavior, whereas the N-6 diet favored birth of daughters.

Influence of dietary phytochemicals and microbiota on colon cancer risk.

Colon cancer is the third most commonly diagnosed type of cancer in the United States. Lifestyle and dietary patterns influence colon cancer risk both positively and negatively. Among the dietary factors, several plant-derived compounds have been found to afford colon cancer protection. These compounds potentially influence all aspects of colonic cellular regulation and develop complex interrelationships with the colonic microbiome. Increasing understanding of the role of microorganisms in determining the colonic environment has led to awareness of this important interrelationship among dietary factors and the microbial population. Plant-derived polyphenols are active mediators of cellular events, target key carcinogenic pathways, and modulate colonic microbial populations. In turn, the colonic microorganisms metabolize dietary compounds and mediate cellular events. In addition, the role of estrogen receptors in colon cancer and the importance of dietary components that mediate estrogen receptor-β are increasingly being discovered. Hence, dietary bioactive compounds and the intestinal microbiota create a complex milieu that directly affects the carcinogenic events of the colon. These relationships must be carefully characterized in future research to provide dietary recommendations that will reduce colon cancer risk.

Aggressive Prostate Cancer Is Prevented in ERαKO Mice and Stimulated in ERβKO TRAMP Mice

Previous evidence suggests soy genistein may be protective against prostate cancer, but whether this protection involves an estrogen receptor (ER)-dependent mechanism is unknown. To test the hypothesis that phytoestrogens may act through ERα or ERβ to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERβ with transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Dietary genistein reduced the incidence of cancer in ER wild-type (WT)/transgenic adenocarcinoma of mouse prostate mice but not in ERα knockout (KO) or ERβKO mice. Cancer incidence was 70% in ERWT mice fed the control diet compared with 47% in ERWT mice fed low-dose genistein (300 mg/kg) and 32% on the high-dose genistein (750 mg/kg). Surprisingly, genistein only affected the well differentiated carcinoma (WDC) incidence but had no effect on poorly differentiated carcinoma (PDC). No dietary effects have been observed in either of the ERKO animals. We observed a very strong genotypic influence on PDC incidence, a protective effect in ERαKO (only 5% developed PDC), compared with 19% in the ERWT, and an increase in the incidence of PDC in ERβKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERβ location or expression. In conclusion, genistein is able to inhibit WDC in the presence of both ERs, but the effect of estrogen signaling on PDC is dominant over any dietary treatment, suggesting that improved differential targeting of ERα vs. ERβ would result in prevention of advanced prostate cancer.

Soy Protein Diet, but Not Lactobacillus rhamnosus GG, Decreases Mucin-1, Trefoil Factor-3, and Tumor Necrosis Factor-α in Colon of Dextran Sodium Sulfate-Treated C57BL/6 Mice

The incidence of inflammatory bowel diseases has increased during recent decades. Within the colon, the families of mucins (MUC) and trefoil factors (TFF) facilitate mucosal protection. Probiotic administration influences the intestinal MUC layer. Additionally, food components may affect gut microflora or have direct effects on the MUC barrier. Our objective was to determine whether diet and/or Lactobacillus rhamnosus GG (LGG) would mediate dextran sodium sulfate (DSS)-induced colitis by altering expression of the MUC and TFF genes. C57BL/6 mice were fed diets containing 20% (wt:wt) casein, soy, or whey proteins with or without LGG for 12 d. Seven days after starting LGG diets, the mice were given 2% DSS in drinking water for 4 d. Two additional casein groups with or without LGG were given tap water, for a total of 8 groups. One day after the DSS treatment, the mice were killed and the colon and cecum tissues and cecum contents were collected and analyzed by qRT-PCR. Whey protein significantly increased cecal LGG content compared with the other diets. In the casein diet groups, MUC1 and TFF-3 expression in colon was significantly induced by DSS independent of LGG. Compared with other DSS-treated groups, soy protein decreased MUC-1 and TFF-3 in the colon. Similarly, soy protein decreased the impact of DSS on inflammatory scores, TNFα gene expression, and colon shortening. There was no overall effect of LGG on these measurements. In conclusion, soy protein suppressed the DSS-induced inflammatory stimulation of MUC, TFF, and TNFα gene expression independently of LGG.

Insulin, IGF-1, and IGF-2 receptors in rat small intestine following massive small bowel resection : analysis by binding, flow cytometry, and immunohistochemistry

This study was undertaken to correlate changes in insulin, IGF-1, and IGF-2 receptors in enterocytes both during the phase of active hyperplasia (protocol 1) and the phase of initiation of hyperplasia (protocol 2) induced by 60% proximal jejunoileal resection in rats. Hormone binding to purified receptor preparations, indirect immunofluorescence analysis by flow cytometry, and immunohistochemistry were used to identify receptor changes. Insulin and IGF-2 receptor binding were increased in the intestine two days after surgery and prior to increased cell mass. The number of cells expressing insulin and IGF-1 receptors increased two-and three-fold between 12 and 36 hr after resection, whereas IGF-2 receptors were maintained throughout the 48-hr period. A significant increase in immunoreactive IGF-2 receptors in both the villus and crypt regions of the jejunum and ileum was observed 12 hr after resection, and this increase was maintained in the crypt region of the jejunum through 48 hr. Therefore, insulin and IGF-2 receptors appear to be important in the initiation of cellular hyperplasia following resection.

Neuropeptide Y fails to normalize food intake in zinc-deficient rats.

Zinc deprivation results in decreased and cyclic food intake in rats. We determined the response of zinc-deprived rats to neuropeptide Y (NPY). In a preliminary experiment, rats were fed a low (-Zn; <1 mg/kg) or adequate zinc diet (+Zn; 100 mg/kg) for 4 days. NPY (5 or 10μg) was then administered via an intracerebroventricular (ICV) cannula and food intake measured for 4h. NPY stimulated food intake in all rats, but the difference in food intake due to zinc deprivation persisted. In a subsequent experiment, rats were fed the low zinc and adequate zinc diets for 4, 5 or 6 days. Food intake was suppressed in rats fed the low zinc compared to the adequate zinc diet on all of these days. When NPY (10 μg) was administered at the onset of the light cycle, the food intake was approximately 2.5-fold greater regardless of dietary zinc status, but the amount of food consumed by rats fed low zinc was approximately one-half the quantity consumed by NPY-stimulated zinc-adequate rats. NPY administered at the onset of dark failed to stimulate food intake in either dietary group although the total intake difference due to zinc status persisted. ICV administration of 5nmol of zinc prior to NPY injection failed to correct the food intake response of the zinc-deficient rats. We conclude that the basis of the reduced food intake of zinc-deficient rats does not relate to NPY quantity or release, or to impairment of its signal transduction. There appears to be another undefined factor that limits food intake in zinc deficiency.