Pear pomace alleviated atopic dermatitis in NC/Nga mice and inhibited LPS-induced inflammation in RAW 264.7 macrophages

BACKGROUND/OBJECTIVESPoorly regulated inflammation is believed to be the most predominant factor that can result in a wide scope of diseases including atopic dermatitis (AD). Despite many studies on the effect of pear pomace in obesity-related disorders including dysregulated gut microbiota, the protective effect of pear pomace in AD is still unknown. This study aimed to evaluate the effect of pear pomace ethanol extract (PPE) on AD by inhibiting inflammation.MATERIALS/METHODSIn the in vivo experiment, 2, 4-dinitrochlorobenzene (DNCB) was applied to NC/Nga mice to induce AD-like skin lesions. After the induction, PPE was administered daily by oral gavage for 4 weeks. The clinical severity score, serum IgE levels, spleen weight, histological changes in dorsal skin, and inflammation-related proteins were measured. In the cell study, RAW 264.7 cells were pretreated with PPE before stimulation with lipopolysaccharide (LPS). Nitrite oxide (NO) production and nuclear factor kappa B (NF-kB) protein expression were detected.RESULTSCompared to the AD control (AD-C) group, IgE levels were dramatically decreased via PPE treatment. PPE significantly reduced scratching behavior, improved skin symptoms, and decreased ear thickness compared to the AD-C group. In addition, PPE inhibited the DNCB-induced expression of inducible nitrite oxide synthase (iNOS), the receptor for advanced glycation end products, extracellular signal-regulated kinase (ERK) 1/2, and NF-κB. PPE inhibited the LPS-induced overproduction of NO and the enhanced expression of iNOS and cyclooxygenase-2. Moreover, the phosphorylation of ERK1/2 and NF-κB in RAW 264.7 cells was suppressed by PPE.CONCLUSIONSThese results suggest that PPE could be explored as a therapeutic agent to prevent AD.     

2,4-Dinitrofluorobenzene-induced contact hypersensitivity response in NC/Nga mice fed fructo-oligosaccharide

Strategies to manipulate gut microbiota in infancy have been considered to prevent the development of allergic diseases later in life. We previously demonstrated that maternal dietary supplementation with fructo-oligosaccharide (FOS) during pregnancy and lactation modulated the composition of gut microbiota and diminished the severity of spontaneously developing atopic dermatitis-like skin lesions in the offspring of NC/Nga mice. The present study tested whether dietary FOS affects contact hypersensitivity (CHS), another model for allergic skin disease, in NC/Nga mice. In experiment 1, 5-wk-old female NC/Nga mice were fed diets either with or without FOS supplementation for 3 wk and then received 2,4-dinitrofluorobenzene (DNFB) on the ear auricle 5 times at 7-d intervals. FOS supplementation reduced CHS response as demonstrated by ear swelling. Quantitative RT-PCR analysis showed that mRNA levels for interleukin (IL)-10, IL-12p40, and IL-17 in the lesional ear skin were significantly lower in mice fed FOS. In experiment 2, female NC/Nga mice were fed diets either with or without FOS during pregnancy and lactation. After weaning, offspring were fed the diets supplemented with or without FOS. Three weeks after weaning, offspring received DNFB on the ear auricle 4 times at 7-d intervals. Although FOS supplementation after weaning reduced ear swelling, maternal FOS consumption was ineffective in offspring. The present data suggest that dietary FOS reduces CHS while maternal FOS consumption is ineffective in offspring of DNFB-treated NC/Nga mice.     

Protease-resistant fraction of smoked, dried bonito alleviates atopic dermatitis-like skin lesions in NC/Nga mice

The effect of smoke-dried bonito undigested fraction remaining after microbial protease treatment (SDBR) on a spontaneously occurring mouse model of atopic dermatitis was studied in male 5-wk-old, NC/Nga mice. Smoke-dried bonito, Katsuobushi, is a traditional Japanese food. SDBR contains 2 major components: bonito oil and protease-undigested proteins. Mice were fed a casein diet containing corn oil (C diet) or a diet containing SDBR (SDBR diet) for 18 wk. In comparison with the C diet, the SDBR diet alleviated the increase in skin severity score and plasma IgE concentration in a time-dependent manner, and lowered leucotriene B(4) (LTB(4))-releasing ability upon calcium ionophore A23187 stimulation. The SDBR diet did not affect scratching time. These results demonstrate that SDBR diet alleviates atopic dermatitis-like skin lesions in NC/Nga mice.     

Oral administration of the extract from Hatakeshimeji (Lyophyllum decastes sing.) mushroom inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice

We examined whether the extract from Hatakeshimeji (Lyophyllum decastes, LD) mushrooms suppresses the development of atopic dermatitis (AD)-like skin lesions induced by repeated application of picryl chloride (PiCl) in NC/Nga mice. Oral administration of LD extract to NC/Nga mice inhibited the development of AD-like skin lesions based on lower total skin severity scores and serum immunoglobulin E (IgE) levels. Splenic lymphocytes were stimulated with the T cell mitogen concanavalin A, and secretion of a Th1 cytokine (IFN-gamma) and a Th2 cytokine (IL-4) was determined by ELISA. IFN-gamma production was not inhibited by treatment with LD extract. On the other hand, IL-4 production was significantly decreased by treatment with LD extract. These results suggest that LD extract exerts anti-allergic actions by suppressing the serum IgE and Th2-type immune responses.     

Genistein suppresses antigen-specific immune responses through competition with 17beta-estradiol for estrogen receptors in ovalbumin-immunized BALB/c mice

OBJECTIVE: The aim of this study was to determine the effects of phytoestrogen genistein on antigen (Ag)-specific immune responses and elucidate the mechanisms underlying those effects. METHODS: Ovalbumin (OVA)-immunized BALB/c mice were administered genistein for 35 d, and OVA-specific immune responses were examined by measuring OVA-specific proliferative responses, production of cytokines, and antibody responses. To assess the effect of genistein on antibody responses to thymus-independent Ag, mice were immunized with 2,4,6-trinitrophenyl (TNP)-Ficoll instead of OVA. Effect of genistein on the functions of CD11c(+) dendritic cells was also examined. Finally, to determine the contribution of estrogen receptor to genistein-mediated immune regulation, mice that had been administered genistein were treated with the estrogen receptor antagonist ICI 182,780 and OVA-specific proliferative responses were examined. RESULTS: OVA-specific proliferative responses and interferon-gamma production levels were decreased in mice administered 20 mg/kg genistein compared with those in control mice without reduction in responses to anti-CD3 monoclonal (m)antibody. The level of OVA-specific immunoglobulin (Ig)G1 was also decreased in mice administered genistein. Levels of OVA-specific IgG2a and IgG2b production and interleukin-4 production in response to OVA were not significantly different but tended to decrease in genistein-treated mice. Genistein administration did not influence the TNP-specific IgM and IgG levels. Furthermore, genistein did not affect the Ag-presenting activity of CD11c(+) dendritic cells. Treatment with ICI 182,780 decreased OVA-specific proliferative responses, but genistein did not suppress these responses synergistically in mice treated with ICI 182,780. CONCLUSIONS: The results of this study suggest that genistein suppresses Ag-specific immune responses. The mechanism underlying the suppression is responsible for the competition of genistein with endogenous 17beta-estradiol for estrogen receptors.     

吴茱萸次碱对硝基氯苯诱导的Nc／Nga小鼠特应性皮炎样皮损的影响

目的观察吴茱萸次碱对特应性皮炎动物模型Nc／Nga小鼠的作用。方法外用2,4-二硝基氯苯（DNcB）反复刺激Nc／Nga小鼠建立特应性皮炎动物模型,通过观察皮肤组织病理学切片、免疫学指标评价皮炎损伤程度,观察吴茱萸次碱对特应性皮炎动物模型Nc／Nga小鼠的治疗作用。结果外用DNCB可以引起Nc／Nga小鼠湿疹样皮炎的产生;模型组小鼠血浆IgE水平（124．42±11．14ng／m1）较正常对照组（17．22±3．56ng／m1）增高（P〈0．05）;治疗组IL-12和IFN-γ水平（62．12±3．14pg／ml,68．29±1．39pg／m1）较模型组血浆IL-12和IFN-γ水平（20．14±6．15pg／ml,51．23±1．45pg／m1）增高,IgE水平（69．17±4．15ng／m1）较模型组IgE水平下降（P〈0．05）;醋酸地塞米松组血浆IL-12、IFN-γ和IgE水平（59．15±1．24pg／ml,64．12±1．19pg／ml,6817±1．15ng／ml）与治疗组比较,差异无统计学意义（P〉0．05）。结论吴茱萸次碱可能通过促进IL-12和IFN-γ分泌,抑制IgE的合成,发挥治疗NdNga小鼠特应性皮炎样皮损的作用。     

Vitamin E Improves Biochemical Indices Associated with Symptoms of Atopic Dermatitis-Like Inflammation in NC/Nga Mice

We aimed to define whether vitamin E improves biochemical indices associated with symptoms of atopic dermatitis-like inflammation in NC/Nga mice. After picryl chloride (PC) application to their backs, changes in the content of thiobarbituric acid reactive substances (TBARS) and vitamin E, as well as the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase) were analyzed in the serum and skin of NC/Nga mice during a symptomatic cycle. The levels of inflammatory factors were also assessed, including IgE, cyclooxigenase-2 (COX-2), tumor necrosis factor (TNF-α) and nuclear factor-κB (NF-κB). When allergic dermatitis was induced by the application of PC to the skin of the mice, skin inflammation appeared 2 wk after PC application, with the peak severity of inflammation observed 5 wk after PC application. Subsequently, the animals recovered from the inflammation by 9 wk after PC application. The TBARS content in the skin and serum increased markedly when the symptoms were the most severe, and decreased to levels near those in control mice by 9 wk after PC application. The activities of SOD and GSHPx in the skin and serum were also positively correlated with symptomatic changes; however, no change in catalase activity was observed 5 wk after PC application. Conversely, vitamin E content decreased at the stage of peak severity. The levels of all inflammatory factors analyzed in this study were altered in a manner similar to other indices. Additionally, vitamin E treatment markedly inhibited these PC-induced alterations. On the basis of these results, it is expected that the observed alterations in biochemical indices, which reflect the symptomatic cycle, may be applicable to objective diagnosis and treatment for atopic dermatitis, and that vitamin E may improve the symptoms of AD.     

Supplemental feeding of a gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid,alleviates spontaneous atopic dermatitis and modulates intestinal microbiota in NC/nga mice

The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer’s patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16?S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.     

Administration of poly(I:C) improved dermatophagoides farinae-induced atopic dermatitis-like skin lesions in NC/Nga mice by the regulation of Th1/Th2 balance

Atopic dermatitis (AD) is characterized by a chronic and replapsing skin disease with Th2-dominant allergic inflammation. Poly(I:C) has been shown to have immunopotentiator properties, but its effect on AD has not been examined. In this study, the immunomodulatory effects of poly(I:C), using dermatophagoides farinae (Df)-induced AD-like skin lesions in NC/Nga mice, were investigated. The clinical scores were reduced significantly by the treatment with poly(I:C) at 25 and 50 μg/mouse. Histological analysis of the skin also revealed that treatment of poly(I:C) at 25 and 50 μg/mouse significantly reduced the inflammatory cellular infiltrate, including mast cells and eosinophils. Moreover, poly(I:C) increased the level of IFN-γ, a Th1 cytokine, whereas decreasing that of selective Th2 cytokine both in vivo and in vitro. The levels of serum IgE and Th2 chemokines such as eotaxin, TARC, in spleen cells were also reduced by poly(I:C). These results suggest that poly(I:C) inhibit the development of Df-induced AD-like skin lesions in NC/Nga mice through regulation of the Th1/Th2 balance. Therefore, our results indicate that poly(I:C) might be a useful immunomodulatory agent for the treatment of human AD.     

Genistein stimulates hematopoiesis and increases survival in irradiated mice

Radiation protection from death and stimulating hematopoietic recovery by oral administrations of genistein, 160 mg/kg b.w., once daily for seven consecutive days before whole-body gamma-rays irradiation, were confirmed by tests with adult male BALB/c mice. Moreover, the protective action of genistein was compared to that of diethylstilbestrol (DES). Based on the studies of survival, behavior of hematograms, endogenous hematopoietic spleen colony formation (endoCFUs), and numbers of nucleated cell, granulocyte-macrophage colony forming units (CFU-GM) in bone marrow following irradiation, it was demonstrated that genistein was an effective radioprotector. The survival of irradiated mice protected by genistein was significantly increased and statistically higher than that of mice pre-treated with DES. Stimulated recovery of leukocytes, erythrocytes, lymphocytes and thrombocytes were observed in mice pre-treated with genistein or DES, however, the effects of genistein on promoting recovery of bone marrow nucleated cells, leukocytes and lymphocytes were significantly higher than those of DES. Enhanced endoCFUs, numbers of bone marrow nucleated cells and CFU-GM were also found in mice pre-treated with genistein as well as DES. Meanwhile, endoCFU numbers in mice pre-treated with genistein was 3.47-fold higher than that in the irradiated control group, although no significant difference was found between genistein administration and DES administration. It could be deduced that the radioprotective action against death is induced by a possible process of enhanced regeneration of the hematopoietic stem cells due to not only strengthened radioresistance and increased numbers of remained hematopoietic cells, but also enhanced post-irradiation repair or promoted proliferation of the hematopoietic stem cells. These effects of genistein may have some therapeutic implications for radiation-induced injuries.     

Lithospermum erythrorhizon Alleviates Atopic Dermatitis-like Skin Lesions by Restoring Immune Balance and Skin Barrier Function in 2.4-Dinitrochlorobenzene-Induced NC/Nga Mice

The incidence of atopic dermatitis (AD), a disease characterized by an abnormal immune balance and skin barrier function, has increased rapidly in developed countries. This study investigated the anti-atopic effect of Lithospermum erythrorhizon (LE) using NC/Nga mice induced by 2,4-dinitrochlorobenzene. LE reduced AD clinical symptoms, including inflammatory cell infiltration, epidermal thickness, ear thickness, and scratching behavior, in the mice. Additionally, LE reduced serum IgE and histamine levels, and restored the T helper (Th) 1/Th2 immune balance through regulation of the IgG1/IgG2a ratio. LE also reduced the levels of AD-related cytokines and chemokines, including interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor-α (TNF-α), thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, regulated on activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 in the serum. Moreover, LE modulated AD-related cytokines and chemokines expressed and secreted by Th1, Th2, Th17, and Th22 cells in the dorsal skin and splenocytes. Furthermore, LE restored skin barrier function by increasing pro-filaggrin gene expression and levels of skin barrier-related proteins filaggrin, involucrin, loricrin, occludin, and zonula occludens-1. These results suggest that LE is a potential therapeutic agent that can alleviate AD by modulating Th1/Th2 immune balance and restoring skin barrier function.     

Genistein enhances antigen-specific cytokine production in female DO11.10 transgenic mice

Genistein is a phytoestrogen contained at high levels in soy products and has been shown to regulate immunoresponse. In this study, we evaluated the effects of genistein on the production of cytokines from antigen (Ag)-specific T cells using DO11.10 transgenic mice because the direct effect of genistein on Ag-specific cytokine production has not been elucidated. The oral administration of 20 mg/kg genistein increased IFN-gamma and IL-4 production from DO11.10+ T cells in response to ovalbumin (OVA)323-339 peptide in female DO11.10 mice. Analysis of intracellular cytokine synthesis revealed that the percentages of cytokine-producing cells in the control and genistein-treated groups were not different, indicating that increased cytokine production occurred at the single-cell level. In contrast to the female mice, genistein did not increase cytokine production in male mice, suggesting that the effect of genistein on cytokine production is gender-dependent.     

Genistein and Daidzein/Glycitein Content in Tofu

The consumption of soyfoods is of great interest because of their proposed anticancer and antiatherogenic activity. It has been suggested that the soy isoflavones genistein and daidzein are responsible for these activities. The objective of this study was to determine the variation of the isoflavone content of commercially available packaged tofu by brands and batches. The content of total genistein and daidzein/glycitein aglycones in tofu samples was determined by high-performance liquid chromatography after acid hydrolysis in 23 different brands and different batches of four of these brands. Between different tofu brands the genistein and daidzein/glycitein content varied from 0.07 to 0.34 mg/g and 0.10 to 0.24 mg/g wet weight, respectively. The isoflavone content from batch to batch of the same brand varied between 7 and 28% based on dry weight. Assuming a daily consumption of 30 g soy products, the isoflavone intake could vary between 5.4 and 17.1 mg/day.     

Citrus auraptene suppresses azoxymethane-induced colonic preneoplastic lesions in C57BL/KsJ-db/db mice

The current study was designed to investigate whether dietary citrus auraptene (AUR) suppresses the development of azoxymethane (AOM)-induced colorectal preneoplastic lesions in C57BL/KsJ-db/db (db/db) mice with obese and diabetic phenotypes. Female db/db and wild (+/+) mice were divided into the AOM + AUR, AOM alone, AUR alone, and the untreated groups in each phenotype. AOM was given 3 weekly intraperitoneal injections (10 mg/kg bw). AUR (250 ppm) was given in diet during the study (for 10 wk). Dietary AUR significantly reduced the number of aberrant crypt foci (ACF) and Beta -catenin-accumulated crypt (BCAC) in both phenotypes. The treatment also lowered cell proliferation activity and increased apoptotic cells in both lesions. Our findings indicate that dietary AUR is able to suppress the early phase of colon carcinogenesis in both phenotypes, suggesting possible application of AUR as a chemopreventive agent in both the high-risk and general populations for colorectal cancer.     

Low-dose ethanol aggravates allergic dermatitis in mice

Alcohol injures dendritic cells and suppresses cellular immunity, while some evidence indicates that drinking alcohol aggravates allergic asthma. This study investigated the effect of low doses of ethanol in enhancing allergic reactions in the skin of mice. Liquid food containing alcohol was administered to conventional NC/Nga mice to induce alcoholic hepatic steatosis, and spontaneous dermatitis was evaluated. BALB/c mice were administered approximately 1 g/kg body weight of ethanol by gavage, and contact hypersensitivity (CHS) or active cutaneous anaphylaxis (ACA) was induced. Spleens were collected 24 h after the elicitation of CHS and mRNA expressions of interferon (IFN)-γ, interleukin (IL)-4, IL-6, IL-10, and IL-18 were measured by quantitative RT-PCR. Alcohol-containing diet exaggerated spontaneous dermatitis in conventional NC/Nga mice and contact hypersensitivity in BALB/c mice. Ethanol administered by gavage for 5 days enhanced contact hypersensitivity in BALB/c mice. Ethanol administration with gavage also enhanced ACA of BALB/c mice. Ethanol did not affect mRNA expression of IFN-γ and IL-4, but did enhance IL-6, IL-10, and IL-18 mRNA expression. Histological evaluation revealed an absence of hepatic steatosis in mice administered ethanol by gavage for 5 days. In ethanol-administered mice, inflamed areas presented as lesions or a local extreme accumulation of mononuclear cells in the epidermis. These findings suggest that ethanol enhances the expression of inflammatory cytokines independently from T helper (Th)1/Th2 cytokine phenotypes, causing abnormalities in the epidermis resulting in exacerbated allergic reactivity.     

Dietary flavones suppresses IgE and Th2 cytokines in OVA-immunized BALB/c mice

Background The flavonoids are a diverse family of chemicals commonly found in fruits and vegetables. Previously, we have shown that the two flavones, chrysin and apigenin could suppress the expression of the high affinity IgE receptor FcεRI in human basophilic KU812 cells. We also demonstrated that dietary apigenin decreased IgE level in C57BL/6N mice sera. Aim of the study To evaluate the anti-allergic effect of the two flavones in vivo, we evaluated the effect of the two flavones, chrysin and apigenin, on the immune system in BALB/c mice sensitized with ovalbumin (OVA). Methods Mice were fed experimental diets containing either of the flavones for 3 weeks and immunized with OVA. After the experimental feeding period, measurement of Igs concentration in the mice sera was performed using a sandwich ELISA. Cytokines expression in mice sera was assessed using a cytokine array. Furthermore, cytokines mRNA levels in spleen lymphocytes from mice sensitized with OVA were measured by RT-PCR. Results The total IgE level in mice fed one of the two flavones were suppressed, whereas levels of IgG, IgM, and IgA were not affected. The production of interleukin (IL)-4, which is known as one of Th2 cytokines and regulates the production of IgE, was down-regulated by the chrysin or the apigenin diet. Moreover, OVA-induced mRNA expression of Th2 cytokines in spleen lymphocytes from mice sensitized with OVA, such as IL-4 and IL-13 were down-regulated by the chrysin or the apigenin diet. Conclusions The results suggest that the diet containing one of the two flavones might suppress the up-regulation of serum IgE induced by OVA-immunization through the suppression of Th2-type immune response.     

Calorie restriction and spontaneous hepatic tumors in C3H/He mice

Caloric restriction started at the young adult (YA) stage and the full adult (FA) stage in mice was compared, specifically focussing on whether there would be a delay in the onset time of spontaneous hepatoma or a reduction in its frequency. Caloric restriction lengthened the life spans of both groups, the YA, and FA. Both groups showed striking reductions of spontaneous hepatomas, from 70.9 +/- 3.5% for non-restricted controls down to 35.7 +/- 5.7 and 30.4 +/- 4.0%, for mice restricted from young adult, and from full adult stages, respectively; further, the numbers of tumor-free mice in the restricted groups increased by 45.7% and 38.5%, respectively, from 11.5%, in the non-restricted control. The cumulative incidences of hepatoma in the caloric restricted groups showed a delayed and lower incidence compared with those of the non-restricted group; a parallel delay might result from a weakened activity in tumor-promotion, whereas a lower frequency, might reflect a possible reduction of target cells for hepatomata development. Both effects can be assumed to have resulted from caloric restriction. When cumulative incidences of small hepatomas were compared between the two restricted groups, restriction started at the young adult stage is assumed to have caused fewer initiation stresses, as well as to have delayed promotion, as clearly evidenced by a flatter curve of incidence with a lower total incidence. Thus, the time at which caloric restriction is started plays a critical role in its subsequent effects.     

三氯乙烯引起BALB/c裸鼠刺激性接触性皮炎作用机制的研究

目的 研究细胞凋亡在三氯乙烯(TCE)引起BALB/c裸鼠皮肤刺激性损伤中的作用.方法 将30只BALB/c裸鼠按随机的原则分成100%TCE、80%TCE、40%TCE、20%TCE、10%TCE、溶剂对照组(橄榄油),TCE背部皮肤染毒1d,应用原位末端标记法检测裸鼠皮肤组织的细胞凋亡,应用免疫组化S-P法检测皮肤上皮组织Caspase-3的表述.结果 免疫组化结果显示各染毒剂量组皮肤组织的凋亡指数和Caspase-3活力的差异有显著性,低剂量组凋亡指数和Caspase-3活力的综合评分分别为(1.84±0.74)和(1.16±0.38),高剂量组分别为(9.00±1.37)和(4.36±0.57),TCE浓度越高,细胞凋亡和Caspase-3的表达水平越高.结论 TCE染毒后短期可引起皮肤组织的细胞凋亡,细胞凋亡在皮肤组织损伤作用的机制中具有重要的意义.     

Effects of soy-derived isoflavones and a high-fat diet on spontaneous mammary tumor development in Tg.NK (MMTV/c-neu) mice

Phytoestrogens such as isoflavonoids and lignans have been postulated as breast cancer protective constituents in soy and whole-grain cereals. We investigated the ability of isoflavones (IFs) and flaxseed to modulate spontaneous mammary tumor development in female heterozygous Tg.NK (MMTV/c-neu) mice. Two different exposure protocols were applied, either from 4 wk of age onward (postweaning) or during gestation and lactation (perinatal). In the postweaning exposure study, mice were fed IFs or flaxseed in a high-fat diet. In addition, flaxseed in a low-fat diet was tested. Postweaning exposure to IFs and flaxseed tended to accelerate the onset of mammary adenocarcinoma development, although tumor burden at necropsy was not changed significantly. Perinatal IF exposure resulted in enhanced mammary gland differentiation, but palpable mammary tumor onset was not affected. However, tumor burden at necropsy in the perinatal exposure study was significantly increased in the medium- and high-IF dose groups. Comparison of both exposure scenarios revealed a strongly accelerated onset of tumor growth after perinatal high-fat diet exposure compared with the low-fat diet. This study shows that breast cancer-modulating effects of phytoestrogens are dependent both on the background diet and on the timing of exposure in the life cycle.