Christine M. Seppanen

The in vivo antioxidant activity of soybean isoflavones in human subjects

This paper presents the in vivo antioxidant activity of soy isoflavones in human subjects determined by the urinary excretion of secondary lipid peroxidation products. Ten healthy women 18-35 years of age consumed a self-selected diet and avoided legumes, whole grains, and isoflavone containing foods. A powdered soy protein isolate was added daily to their diet that provided 3 levels of isoflavones: control 0.15, low 1.01, and high 2.01 mg/kg body weight. Subjects were randomized to consume all three diets for 13 weeks each, with each subject serving as her own control. Urine samples were analyzed from 24-hr collections at the end of each diet period for lipophilic aldehydes and related carbonyl compounds by HPLC. Results show that six of the individual urinary nonpolar compounds (NPC) levels were significantly lower due to consumption of the high isoflavone diet and one was also significantly lower due to consumption of the low isoflavone diet. The total of the individually measured urinary NPC was significantly lower with consumption of both the low and high isoflavone diets when compared with the control diet.

Combination of Intermittent Calorie Restriction and Eicosapentaenoic Acid for Inhibition of Mammary Tumors

There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines. Cancer Prev Res; 6(6); 540–7. ©2013 AACR.

The effect of paprika carotenoids on in vivo lipid peroxidation measured by urinary excretion of secondary oxidation products

Abstract The in vivo antioxidant effects of high levels of dietary paprika carotenoids were investigated by comparison to a high level of β-carotene and to a physiological level of vitamin E. Dietary (vitamin E deficiency) and chemical (CCl 4 ) treatments were used to produce lipid peroxidation in vivo in order to demonstrate the antioxidant effects of paprika carotenoids and β-carotene, which were determined by the urinary excretion of aldehydic secondary lipid oxidation products. Rats were fed a diet which contained either a normal level (0.003%) of vitamin E as RRR-α-tocopherol acetate (NE), a vitamin E deficient diet (−E), or a vitamin E deficient diet supplemented with 0.5% or 1.0% paprika carotenoids (−E + PCI or −E + PCII), or 0.5% β-carotene (−E + BC) for 18 weeks. One group of animals on the NE diet was also stressed with CCl 4 (NE + CCl 4 ). Urine samples were separated by high performance liquid chromatography (HPLC) into six fractions which contained free and conjugated malondialdehyde (MDA) and four groups of other secondary aldehydic lipid peroxidation products. Results show that the antioxidant treated groups, NE, −E + BC, −E + PCI, and −E + PCII, all had lower levels of urinary excretion of free and conjugated MDA, non-MDA carbonyl compounds, and excretion of all (MDA and non-MDA) aldehydic secondary lipid oxidation products than the oxidatively stressed −E and NE + CCl 4 groups. Rats fed the NE diet, however, had significantly higher body weights at 18 weeks than the other groups. The results indicate that dietary paprika carotenoids and β-carotene, when fed at high levels, exhibit similar antioxidant effects on inhibiting in vivo lipid peroxidation but do not compensate for the role of physiological levels of vitamin E in normal growth and weight gain.

Effect of high stearic acid containing fat on markers for in vivo lipid peroxidation

The objective of this study was to determine the effect of high stearic acid (SA) diets versus high polyunsaturated fatty acid (PUFA) diets on several measures of lipid peroxidation in vivo. Sprague–Dawley rats were fed diets that differed only in the fat source (8% by weight) for 19 weeks. High SA fats were beef tallow (BT) and cocoa butter (CB), high PUFA fats were soybean oil (SO) and menhaden oil (MO). Urine was analyzed for lipophilic aldehydes, the secondary products of lipid peroxidation, by HPLC. Decreases (P<0.05) were found for 4 nonpolar lipophilic aldehydes and related carbonyl compounds (NPC) and 4 polar lipophilic aldehydes and related carbonyl compounds (PC) when the BT-fed group was compared to the SO-fed group. Decreases were also found to be significant for total NPC (P<0.01) and total PC (P<0.05) between BT and SO-fed groups. Serum increase in resistance to oxidation (P<0.01) was found in the BT group when compared to the SO group. The differences in urine and serum measurements in the present experiment indicate lower level of lipid peroxidation in vivo due to the consumption of high SA containing BT diet compared to high PUFA containing SO diet without raising serum triglycerides and cholesterol levels significantly for the BT-fed groups.

Abstract 585: Eicosapentaenoic acid increases mammary tumor inhibition of intermittent calorie restriction and regulates adipokines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It has become clear that the use of combination therapies for prevention of breast cancer may be more efficacious than individual therapies. Therefore, we combined two different interventions which had each been shown alone to inhibit mammary tumorigenesis but had not been tested in combination. The first intervention was consumption of the omega-3 fatty acid eicosapentaenoic acid (EPA) which is found in fish oil and the second intervention was calorie restriction. We utilized six groups of MMTV-Her2/neu mice as a model for human breast cancers which over express Her2/neu. Starting at 10 weeks of age half of the mice were fed a control diet composed of a modified AIN-93M formula with fat derived from soy oil. The other half consumed a diet with 71.75% of fat calories from EPA. Mice were further divided into ad libitum (AL), chronic calorie restricted (CCR) or intermittent caloric restricted (ICR) groups. AL groups (Control and EPA) received unrestricted access to their diets. CCR groups were given 75% of the total calories that the AL age-matched groups consumed. ICR groups were fed calories equal to 100% of the AL age-matched groups for three weeks followed by three weeks of calories equal to 50% of the AL age-matched groups. The 6 week cycles of restriction and refeeding were maintained until the mice were 60 weeks of age or were euthanized due to mammary tumors (MTs). MT incidence was lowest in the ICR-EPA group (15%) and highest in the AL-Con group (87%) while the AL-EPA, CCR-Con, CCR-EPA and ICR-Con had MT incidence rates of 63%, 47%, 40% and 59%, respectively The average survival until terminal end point was also longest in the ICR-EPA group at 59.2 weeks and shortest in the AL-Con group at 52.6 (P<0.0002). The average tumor burden was 0.98 grams for ICR-EPA mice and 1.78 grams for AL-Con mice but due to the very small number of MTs in the ICR-EPA group this was not a significant difference. Histopathology showed no significant difference in tumor grades between the groups. To better understand the effects of the interventions we examined serum for alterations in adipokines and growth factors. We found that EPA consumption dramatically reduced the levels of leptin (P<0.02) and increased the levels of adiponectin in the AL mice (P<0.001) but did not significantly alter animal weights at 25 weeks of age. Calorie restriction was also able to significantly decrease serum leptin levels and increase serum adiponectin levels. Serum IGF-I was significantly reduced by calorie restriction with the ICR-Con having the lowest level. The presence of EPA in the diet did not significantly change the levels of IGF-I. These results illustrate that MT inhibition is significantly increased when ICR and EPA are combined as compared to either intervention alone or no intervention. This effect may be related to alterations in serum growth factors and adipokines. Support: Susan G.Komen for the Cure and The Hormel Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 585. doi:1538-7445.AM2012-585

Abstract 597: Response of reactive oxygen species to eicosapentaenoic and calorie restriction in relation to mammary tumor prevention

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Exposure of lipid biomembranes to reactive oxygen species (ROS) results in the generation of a wide variety of lipid peroxidation products. Some of these products, including 4-hydroxy-2-nonenal (4HNE) can form protein adducts which may impact pathways associated with tumorigenesis. We have reported that calorie restriction protects against spontaneous mammary tumor development in mice and have recently shown that this is accompanied by a decrease in serum levels of lipid peroxidation products. Eicosapentaenoic acid (EPA) is known to inhibit mammary tumorigenesis; however, this type of highly unsaturated omega-3 fatty acid has also been shown to increase in vivo levels of lipid peroxidation products. In the present study, the interaction of calorie restriction and dietary EPA was investigated with respect to mammary tumor formation. Lipid peroxidation products of ROS were also measured. From 10 weeks of age MMTV-Her2/neu mice were fed a modified AIN-93M diet (10.2% kcal from fat) which had either 100% soybean oil (SO) as the fat source or a blend of EPA (72%) and SO (18%). Mice were further divided into groups which were ad libitum-fed, (AL); chronic calorie restricted (CCR, received 75% of AL) or intermittently calorie restricted (ICR, received 50% of AL for 3 weeks followed by 3 weeks at 100% of AL) until 60 weeks of age or until mammary tumor size required euthanasia. Mice were weighed and examined for mammary tumors weekly. Serum thiobarbituric acid reactive substances (TBARS) and 4-hydroxy-2-nonenal (4HNE)-protein adducts in mammary tissues were measured to assess the extent of lipid peroxidation. In both diet groups, mammary tumor incidence was highest in the AL group (SO 87%, EPA 63%). These groups also had the highest levels of serum TBARS and the AL-SO group had the highest level of mammary tissue 4HNE-protein adducts. The calorie restricted mice had lower levels of mammary tumor incidence (CCR-SO 47%, CCR-EPA 40%, ICR-SO 59%) with the lowest level in the ICR-EPA group (15%). The ICR-SO group had serum TBARS and mammary tissue 4HNE-protein adduct levels that were significantly lower than the AL-SO group (p<0.05 for both). In the EPA groups, there was no difference between the AL, CCR, and ICR groups in either serum TBARS or mammary tissue 4HNE-protein adducts. These results suggest that higher levels of lipid peroxidation products are associated with a higher incidence of mammary tumors. However, although the dietary combination of ICR and EPA resulted in the greatest amount of mammary tumor inhibition, this combination was not associated with the lowest level of lipid peroxidation products. The relationship between EPA, calorie restriction, and reactive oxygen species appears to be quite complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 597. doi:1538-7445.AM2012-597

Abstract 1869: Mammary tumor inhibition by intermittent calorie restriction and eicosapentaenoic acid

Breast cancer is a complex disease and research as well as compliance may be more productive if combinations of dietary intervention strategies are utilized. For this study we combined consumption of the omega-3 fatty acid eicosapentaenoic acid (EPA) with two modes of calorie restriction. We utilized six groups of MMTV-Her2/neu mice to represent the approximately 20% of human breast cancers in which Her2/neu is over expressed and for which there are no known chemo-preventive agents available. Starting at 10 weeks of age half of the mice were fed a control diet composed of a modified AIN-93M formula with fat derived from soy oil. The other half consumed a diet with 71.75% of fat calories from EPA. Mice were further divided into ad libitum (AL), chronic calorie restricted (CCR) or intermittent caloric restricted (ICR) groups. AL groups (Control and EPA) received unrestricted access to their diets. CCR groups were given 75% of the total calories that the AL age-matched groups consumed. ICR groups were fed calories equal to 100% of the AL age-matched groups for three weeks followed by three weeks of calories equal to 50% of the AL age-matched groups. Supplemental vitamins and minerals were added to the food for the CCR ands ICR groups. The 6 week cycles of restriction and refeeding were maintained until the mice were 60 weeks of age or were euthanized due to MTs. Mice were weighed weekly and carefully examined for mammary tumors (MTs). Ad Lib Control (Con) and Ad Lib-EPA groups ate similar amounts and gained weight at similar rates. The body weights of the CCR-Con and CCR- EPA were also similar to each other. The ICR-EPA mice were slightly heavier (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2011-1869

Abstract B63: Combination of intermittent calorie restriction and eicosapentaenoic acid for inhibition of mammary tumors

Introduction: Nutrition can have a profound effect on breast cancer inhibition and growth. We have focused on two dietary interventions that separately prevent mammary tumorigenesis, eicosapentaenoic acid (EPA), an ω-3 fatty acid, and intermittent calorie restriction (ICR), as we recently reported that multiple periods of intermittent caloric restriction (ICR) were superior for inhibition of mammary tumor (MT) formation compared to chronic calorie restriction. Here we assessed whether combining two interventions, EPA consumption and ICR, would achieve even greater inhibition of breast cancer formation than either alone. Methods: Four groups of MMTV-HER-2/neu transgenic mice that are genetically programmed to develop mammary tumors were utilized. From 10 weeks of age half of the mice were fed a diet with fat derived from soy oil and the other half consumed a diet with 71.75% of fat calories from EPA. Mice were further divided into ad libitum (AL) or intermittent caloric restricted (ICR) groups. AL groups (soy oil and EPA) received unrestricted access to their diets. ICR groups were fed calories equal to 100% of the AL age-matched groups for three weeks followed by three weeks of calories equal to 50% of the AL age-matched groups. Mice were weighed weekly and carefully examined for mammary tumors. The 6 week cycle of restriction/refeeding was maintained until the mice were 55 weeks or were euthanized due to MTs. Results: The Ad Lib Soy and Ad Lib EPA groups ate similar amounts and gained weight at similar rates. The body weights of the ICR Soy and ICR EPA groups were not significantly different from each other but were significantly lower than the Ad Lib mice. The tumor free period was shortest in the AL Soy with increasingly long tumor free periods for the Ad Lib EPA, ICR Soy and the ICR EPA groups. The tumor free period of the ICR EPA group was significantly longer than all the other groups (ICR EPA vs Ad Lib Soy P Conclusions: These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared to either intervention alone or no intervention. It also identifies the NFκB pathway as a potential pathway of interest for inhibition of mammary tumor formation. Understanding how this pathway is affected may aid in the development of drugs that could be used for breast cancer prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B63.