Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper.

PURPOSE: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. EXPERIMENTAL DESIGN: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. RESULTS: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. CONCLUSIONS: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

Pharmacological inhibition of MERTK induces in vivo retinal degeneration: a multimodal imaging ocular safety assessment.

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Aggressive prostate cancer is prevented in ERαKO mice and stimulated in ERßKO 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 ERß to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERß 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 ERßKO 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 ERßKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERß 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. ERß would result in prevention of advanced prostate cancer.

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-ß 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.

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.

Common botanical compounds inhibit the hedgehog signaling pathway in prostate cancer.

Many botanical compounds have been proposed to prevent cancer. We investigated the cancer treatment and prevention abilities of apigenin, baicalein, curcumin, epigallocatechin 3-gallate (EGCG), genistein, quercetin, and resveratrol both in vivo in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice as well as in vitro in prostate cancer cell lines. In our experiments, these seven compounds act similarly to the Hedgehog antagonist cyclopamine, a teratogenic plant alkaloid, which had been previously shown to "cure" prostate cancer in a mouse xenograft model. With IC(50) values ranging from <1 to 25 mumol/L, these compounds can inhibit Gli1 mRNA concentration by up to 95% and downregulate Gli reporter activity by 80%. We show that four compounds, genistein, curcumin, EGCG, and resveratrol, inhibit Hedgehog signaling as monitored by real-time reverse transcription-PCR analysis of Gli1 mRNA concentration or by Gli reporter activity. Three compounds, apigenin, baicalein, and quercetin, decreased Gli1 mRNA concentration but not Gli reporter activity. Our results show that these compounds are also able to reduce or delay prostate cancer in vivo in TRAMP mice. All seven compounds, when fed in combination as pure compounds or as crude plant extracts, inhibit well-differentiated carcinoma of the prostate by 58% and 81%, respectively. In vitro, we show that all seven compounds also inhibit growth in human and mouse prostate cancer cell lines. Mechanistically, we propose the Hedgehog signaling pathway to be a direct or indirect target of these compounds. These botanicals at pharmacologic concentrations are potentially safer and less expensive alternatives to cyclopamine and its pharmaceutical analogues for cancer therapy.

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, RARbeta2, 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 beta 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 RARbeta2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.

Genistein affects HER2 protein concentration, activation, and promoter regulation in BT-474 human breast cancer cells.

The HER2 proto-oncogene, a member of the epidermal growth factor receptor family, is overexpressed in 20-30% of breast cancers. Genistein, the main soy isoflavone, interacts with estrogen receptors (ER) and it is also a potent tyrosine kinase inhibitor. Previously, our laboratory found that genistein delayed mammary tumor onset in transgenic mice that overexpress HER2 gene. Our goal was to define the mechanism through which genistein affects mammary tumorigenesis in HER2 overexpressing mice. We hypothesized that genistein inhibits HER2 activation and expression through ER-dependent and ER-independent mechanisms. Genistein inhibited total HER2 protein expression and tyrosine phosphorylation in BT-474, an ERalpha (-) and ERbeta (+) human breast cancer cell line, however, E2 had no effect. Taken together, these data suggest that genistein has an ER-independent inhibitory effect, presumably, through tyrosine kinase inhibition activity. Genistein at 1.0 microM mimicked E2 and down-regulated HER2 protein phosphorylation when BT-474 was co-transfected with ERalpha, but not ERbeta. Although E2 and overexpression of HER2 can promote mammary tumorigenesis, an inverse relationship between ER expression and HER2 overexpression has been found in human breast cancer. We cloned a 500-bp promoter region upstream of the HER2 transcription initiation site. Co-transfection with ERalpha, but not with ERbeta, down-regulated HER2 promoter reporter in BT-474. At concentrations > or =1 microM, genistein inhibited HER2 promoter reporter in the absence of ERalpha. In conclusion, genistein at > or =1 microM inhibited HER2 protein expression, phosphorylation, and promoter activity through an ER-independent mechanism. In the presence of ERalpha, genistein mimicked E2 and inhibited HER2 protein phosphorylation. These data support genistein's chemo-prevention and potential chemo-therapeutic roles in breast cancer.

Phytosterol Pygeum africanum regulates prostate cancer in vitro and in vivo.

BACKGROUND: Prostate cancer is an important public health problem. It is an excellent candidate disease for chemoprevention because prostate cancer is typically slow growing and is usually diagnosed in elderly males. Pygeum africanum (Prunus africana or Rosaceae) is an African prune (plum) tree found in tropical Africa. An extract from the bark of Pygeum africanum has been used in Europe as a prevention and treatment of prostate disorders including benign prostatic hypertrophy (BPH). More recently in the USA, the phytotherapeutic preparations of Pygeum africanum and Saw palmetto have been marketed for prostate health including prostate cancer prevention and treatment. METHODS: The anti-cancer potential of Pygeum africanum has been tested both in vitro (PC-3 and LNCaP cells) and in vivo (TRAMP mouse model). RESULTS: In tissue culture, ethanolic extracts (30%) of Pygeum africanum inhibited the growth of PC-3 and LNCaP cells; induced apoptosis and altered cell kinetics; down regulated ERalpha and PKC-alpha protein, and demonstrated good binding ability to both mouse uterine estrogen receptors and LNCaP human androgen receptors. TRAMP mice fed Pygeum africanum showed a significant reduction (P = 0.034) in prostate cancer incidence (35%) compared to casein fed mice (62.5%). CONCLUSION: Pygeum africanum, which is widely used in Europe and USA for treatment of BPH, has a significant role in regulation of prostate cancer both in vitro and in vivo and therefore may be a useful supplement for people at high risk for developing prostate cancer.

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.

Environmental influences on isoflavones and saponins in soybeans and their role in colon cancer.

Soybeans have long been recognized as an excellent source of high-quality protein. The soybean also contains a wide variety of chemical compounds that have potent bioactivity. Among these compounds are the isoflavones and the saponins. The goal of our research was to quantify isoflavone and saponin concentrations in elite soybean cultivars grown in different environments and to identify a naturally occurring high and low variety that could be used in animal studies of colon cancer. We observed significant environment x genotype interactions for the cultivars and selected 2 that provided the range of concentration for isoflavones and saponins. These were grown in an adequate quantity for animal studies, which are ongoing. We explored the influence of isoflavones and saponins on human colon tumor cells in culture, Caco-2, to determine potential mechanisms through which these compounds influence the carcinogenic process. We observed the inhibition of Caco-2 cell proliferation by isoflavones and saponins, suggesting a protective effect of these compounds in colon cancer. Using purified soy saponins, we found no negative effects on mouse growth, organ weights, or intestinal morphology when the diet contained up to 3% saponins by weight. Hence, soy isoflavones and saponins are likely to be protective of colon cancer and to be well tolerated. Continuing studies will explore the cancer-protective effects of these compounds in animal models.

Dietary grape supplement ameliorates cerebral ischemia-induced neuronal death in gerbils.

Oxidative damage has been implicated as one of the leading causes for neuronal cell death in a number of neurodegenerative diseases including stroke. Many vegetables and fruits are enriched in polyphenolic compounds known to exhibit antioxidant properties. This study is to investigate whether dietary supplement with grape powder (GP) may offer protection against neuronal damage due to global cerebral ischemia induced to Mongolian gerbils by occlusion of the common carotid arteries, a model known to cause delayed neuronal death (DND) in the hippocampal CA1 area. Gerbils were fed either a control diet (AIN76a) or a control diet supplemented with low (5.0 g/kg diet) or high (50 g/kg diet) levels of GP for two months. Four days after ischemia/reperfusion (I/R), the extent of DND, glial cell activation, nuclear DNA oxidation, and apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction in hippocampal CA1 region were assessed. Ischemia-induced extensive DND in the CA1 region was accompanied by oxidative and fragmented DNA damage and a marked increase in reactive astrocytes and microglial cells. Dietary GP supplementation significantly protected neurons against I/R-induced DND, DNA damage, and apoptosis as well as attenuated glial cell activation. These results demonstrate that due to the antioxidant properties of polyphenols in GP, nutritional diets supplemented with grape can protect the brain against ischemic damage. The neuroprotective effects of GP supplement may have wide implication in the future for prevention/protection against other neurodegenerative damage.

Phytoestrogens in common herbs regulate prostate cancer cell growth in vitro.

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 M 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.

Dietary soy isoflavones and estrone protect ovariectomized ERalphaKO and wild-type mice from carcinogen-induced colon cancer.

Consumption of soy foods has been weakly associated with reduced colon cancer risk. Colon cancer risk is influenced by estrogen exposure, although the mechanism through which this occurs is not defined. Conversion of estradiol (E2) to estrone (E1) may be protective in the colon. We hypothesized that dietary phytoestrogens, or E1, would reduce colon tumorigenesis via an estrogen receptor (ER)-dependent mechanism. Ovariectomized ERalphaKO or wild-type (WT) female mice were fed diets containing casein (Casein), soy protein without isoflavones (Soy-IF), soy protein + genistein (Soy+Gen), soy protein + NovaSoy (Soy+NSoy) or soy protein + estrone (Soy+E1) from weaning. Colon tumors were induced with azoxymethane. Tumor incidence was affected by diet but not genotype. Colon tumor incidence was lower in ERalphaKO and WT mice fed the Soy+E1 diet compared with those fed the casein or Soy-IF diets. Mice fed Soy+NSoy had a lower tumor incidence than mice fed casein, but not Soy-IF. Genistein did not affect tumor incidence. Soy protein, independently of phytoestrogens or E1, significantly reduced relative colon weight, tumor burden and multiplicity. Relative colon weight was lower (P=0.008) in mice fed Soy+E1 than in the other soy-fed groups. Tumor incidence in this group was lower than in the casein and soy-IF-fed groups and tended to be lower than in the others (P=0.020). Hence, soy protein and NSoy protect mice from colon cancer, and E1 further reduces colon tumorigenesis in mice, independently of ERalpha.

Soy isoflavones increase latency of spontaneous mammary tumors in mice.

Soy protein, with and without isoflavones, is being added to foods by manufacturers in response to the Food and Drug Administration (FDA)-approved health claim for cardiovascular protection. Furthermore, soy isoflavones are increasingly consumed by women in the United States as an alternative to hormone replacement therapy. The role of these phytoestrogens in breast cancer is controversial. Although exposure of rodents to soy isoflavones during the perinatal period appears to reduce mammary cancer formation, exposure in utero or during adulthood may increase tumor growth. The mouse mammary tumor virus (MMTV)-neu mouse spontaneously develops mammary tumors due to overexpression of the ErbB-2/neu/HER2 oncogene. This model is comparable with human breast cancer because overexpression of the neu oncogene occurs in 20-40% of human breast cancers. We fed MMTV-neu mice AIN-93G diets containing no isoflavones, 250 mg/kg genistein, 250 mg/kg daidzein or an isoflavone mixture (NovaSoy, equivalent to 250 mg genistein/kg) from 7 wk of age. Mammary tumor latency was significantly delayed in mice fed isoflavones compared with the control. Once tumors formed, however, the isoflavones did not reduce the number or size of tumors such that at 34 wk of age there were no differences in tumor burden among the treatment groups. Hence, in the MMTV-neu mouse, soy isoflavones delayed mammary tumorigenesis. Further studies are warranted to define the cellular mechanisms through which these compounds affect mammary tumorigenesis in this model.

Genistein alters methylation patterns in mice.

In this study we examine the effect of the phytoestrogen genistein on DNA methylation. DNA methylation is thought to inhibit transcription of genes by regulating alterations in chromatin structure. Estrogenic compounds have been reported to regulate DNA methylation in a small number of studies. Additionally, phytoestrogens are believed to affect progression of some human diseases, such as estrogen-dependent cancers, osteoporosis and cardiovascular disease. Specifically, our working hypothesis is that certain soy phytoestrogens, such as genistein, may be involved in preventing the development of certain prostate and mammary cancers by maintaining a protective DNA methylation profile. The objective of the present study is to use mouse differential methylation hybridization (DMH) arrays to test for changes in the methylation status of the cytosine guanine dinucleotide (CpG) islands in the mouse genome by examining how these methylation patterns are affected by genistein. Male mice were fed a casein-based diet (control) or the same diet containing 300 mg genistein/kg according to one of four regimens: control diet for 4 wk, genistein diet for 4 wk, control diet for 2 wk followed by genistein diet for 2 wk and genistein diet for 2 wk followed by control diet for 2 wk. DNA from liver, brain and prostate were then screened with DMH arrays. Clones with methylation differences were sequenced and compared with known sequences. In conclusion, consumption of genistein diet was positively correlated with changes in prostate DNA methylation at CpG islands of specific mouse genes.

Megestrol acetate increases short-term food intake in zinc-deficient rats.

Rats offered a zinc-deficient (-Zn) diet voluntarily reduce their food intake within 3-4 days. Megestrol acetate (MA) is an appetite-stimulating drug used to treat cachexia of chronic diseases. In previous work, we found MA administration to male rats increased consumption of a -Zn diet. This approach would provide a useful tool for nutritional studies in which nutrient intake, except for zinc, would be maintained. The present study further examined the use of MA to increase consumption of a -Zn diet over a longer time period in both male and female rats. Rats were fed either a -Zn or a zinc-adequate (+Zn) diet. In Experiment 1, rats were treated orally with 0, 20, 50 or 100 mg MA/kg BW in corn oil for 21 days. MA stimulated intake of the -Zn diet in a linear manner. In Experiments 2 and 3, male and female rats, respectively, were fed the -Zn or +Zn diets and treated with 100 mg MA/kg BW for 21 days. In both experiments, MA administration increased intake of the -Zn diet to levels similar to the +Zn diet through Day 14. MA increased the hypothalamic neuropeptide Y (NPY) concentration in male rats, but did not affect serum IGF-I. MA administration improved growth of female but not male rats fed the -Zn diet. In females, serum IGF-I was not lower in zinc-deficient rats, which may have allowed the improved growth response with MA. Hence, MA administration may be a useful tool to increase consumption of a -Zn diet in short-term studies.