Soy protein peptides regulate cholesterol homeostasis in Hep G2 cells.

The activation of LDL receptors was described recently in a human hepatoma cell line (Hep G2) exposed both to alpha + alpha' subunits from 7S soy globulin and to Croksoy(R)70, a commercial isoflavone-poor soy concentrate. To assess the final identity of the peptide(s) putatively responsible for the biochemical effect, experiments were performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin or to peptides from the in vitro digestion of Croksoy(R)70. Moreover, the ability of the whole 7S globulin, its subunits and whole Croksoy(R)70 to interfere in the apolipoprotein B (apo B) secretion in the medium as well as in sterol biosynthesis was evaluated in the same model. Increased (125)I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10(-)(4) mol/L, MW 2271 Da) corresponding to positions 127-150 of the 7S globulin. Cells exposed to Croksoy(R)70 enzyme digestion products showed a more marked up-regulation of LDL receptors vs. controls, compared with vs. Hep G2 cells incubated with undigested Croksoy(R)70. Among soy-derived products, only the 7S globulin inhibited apo B secretion and (14)C-acetate incorporation when tested in Hep G2 cells at a concentration of 1.0 g/L. These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Moreover, the protein moiety, devoid of isoflavone components, is likely to be responsible for this major biochemical effect of soy protein.

Iron status and resting immune function in female collegiate swimmers.

Iron deficiency may lead to anemia and may result in compromised endurance exercise performance. Iron deficiency has also been reported to adversely affect the immune system and has been associated with attenuation of natural killer cell (NK) activity. This study was conducted to examine the relationship between iron status and NK activity in highly conditioned female athletes. Ten collegiate female swimmers (SWM) and 9 inactive females (SED) participated in this investigation. Resting blood samples were obtained and analyzed for serum iron and ferritin. NK activity (% lysis) was determined using a whole blood method (51Cr release assay). No significant relationship was found between iron and NK activity (r = 0.55, p =.09), nor between serum ferritin and NK activity (r = 0.33, p =.35) for SWM. ANOVA revealed significantly greater NK activity for SWM (51.63 +/- 15.79%) versus SED (30.34 +/- 13.67%). Serum ferritin levels were not significantly different between SWM (20.38 +/- 8.62 hg á ml-1) and SED (16.79 +/- 10.53 hg á ml-1), nor were iron values different between groups (16.54 +/- 2. 17 mmol á L-1 SWM; 11.92 +/- 2.61 mmol á L-1 SED). A significant relationship between iron status and resting immune function could not be established. Exercise training may affect NK activity; however, the influence of iron status on immune function requires further evaluation.

Effects of intensified training and detraining on testicular function.

OBJECTIVE: To examine the effects of an increased training load and period of detraining on testicular function in male distance runners. DESIGN: Multiple-group time-series design using a control group. SETTING: University of Toledo and Toledo Hospital. PARTICIPANTS: Eight male runners and eight age-matched sedentary control subjects. Subjects were considered fit for participation after a physical and genital examination conducted by a physician. INTERVENTION: Subjects provided blood and semen samples every 2 weeks for 8 weeks. The training regimen for the runners consisted of 2 weeks at normal training (NT), 2 weeks at 143% of NT (IT1), 2 weeks at 186% of NT (IT2), and 2 weeks at 50% of NT (RT). These percentages represent increases in training distance (volume). MAIN OUTCOME MEASURES: Within the context of this investigation, the following hypothesis was developed: increases or decreases in training would not significantly alter sperm count, density, motility, or morphology, or concentrations of reproductive hormones or cortisol in runners. RESULTS: There were no statistically significant differences observed between runners and control subjects for any of the reproductive hormones or cortisol. In addition, there was no significant treatment effect for sperm count, motility, or morphology. The sperm levels in two runners in this investigation dropped to oligospermatic levels after IT2; however, total sperm count increased in both runners after 2 weeks of RT. CONCLUSION: Four weeks of increased training and 2 weeks of reduced training did not significantly affect the subjects in this investigation. It is possible that a particular level or degree of training must be surpassed before any clinical alterations are evident. Future longitudinal studies are necessary to identify the extent to which endurance training may alter reproductive hormones and testicular function.

Addition of arginine but not glycine to lysine plus methionine-enriched diets modulates serum cholesterol and liver phospholipids in rabbits.

Previous experiments from our laboratory showed that in rabbits fed an amino acid diet corresponding to 30% casein, enrichment of the diet with L-lysine and L-methionine caused a marked increase in serum total and LDL cholesterol levels as well as a substantial body weight loss. Both effects were partially prevented by supplementation with L-arginine. The present studies were designed to extend this earlier observation by assessing the role of different dietary amino acids in modulation of cholesterolemic responses and body weights. In the first experiment, the original lysine and methionine-enriched diet was supplemented with glycine in an attempt to modify methionine metabolism, and thus to reduce body weight loss. In addition, the mechanism of action of lysine and methionine was investigated by quantitation of major liver phospholipids. The results showed that glycine addition had no effect on weight loss or hypercholesterolemia, nor did it alter plasma levels of homocyst(e)ine, an intermediate in methionine metabolism. However, enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus may explain the hypercholesterolemia. A second experiment showed that similar effects on body weights and serum cholesterol could be obtained by adding lysine and methionine to a diet containing amino acids equivalent to only 15% casein, or 15% intact casein. This approach is more physiologic and also reduces the expense of experiments designed to study the effects of lysine and methionine in more detail.

Regulation of HepG2 cell apolipoprotein B metabolism by the citrus flavanones hesperetin and naringenin.

Our previous studies showed that replacing the drinking water of rabbits fed a casein-containing diet with either orange juice or grapefruit juice reduced serum low density lipoprotein cholesterol and hepatic cholesteryl ester concentrations. To determine whether the changes observed in rabbits were due to flavonoids present in the juices acting directly on the liver, the effects of hesperetin and naringenin on net apolipoprotein B (apoB) secretion by HepG2 cells were investigated. These flavanones dose-dependently reduced net apoB secretion by up to 81% after a 24 h incubation, while doses of 60 micrograms/mL reduced net apoB secretion by 50% after 4 h. Coincubation with the proteasome inhibitor, MG-132, did not alter the ability of the flavonoids to reduce net apoB secretion over 4 h, suggesting that the flavonoid-induced changes in apoB metabolism were not due to a direct increase in proteasomal activity. However, the flavonoids were unable to reduce net apoB secretion after 4 h in the presence of oleate, suggesting that these compounds may interfere with the availability of neutral lipids for lipoprotein assembly. Furthermore, our 14C-acetate-labeling studies showed a 50% reduction in cholesteryl ester synthesis in the presence of either flavonoid, which could account for the reduction in net apoB secretion caused by incubation with these compounds. These in vitro studies suggest that hesperetin and naringenin may, in part, reduce net apoB secretion by HepG2 cells by inhibiting cholesteryl ester synthesis and that these compounds are good candidates for further in vivo studies to determine whether they are responsible for the cholesterol-lowering properties of dietary citrus juices.

Specific versus non-specific effects of dietary fat on carcinogenesis.

It will be apparent from this review that dietary fat can exert both specific and non-specific effects on carcinogenesis, at least in experimental animals. The non-specific effects appear to be related primarily to effects of dietary fat on energy balance. Although a positive energy balance can be achieved on a high-carbohydrate low-fat diet, it is much more likely to occur on a high-fat diet because of the high energy density of fat [101] and the fact that dietary fat is less capable of imparting a sense of satiety [102]. A continuing state of positive energy balance leads to obesity which has been associated with increased risk of cancer at a number of sites, including endometrium [103-106], postmenopausal breast cancer [107-113], renal cancer [114,115] and possibly cancers of the colorectum [116-122], pancreas [103,123] and prostate [124]. Whereas the non-specific effects of dietary fat appear to be deleterious for cancer, the specific effects in some cases can be beneficial. Examples are long-chain n-3 polyunsaturated fatty acids. CLA and tocotrienols. It is still too early to predict whether these may be of value in the prevention and/or treatment of human cancer but they seem worthy of further investigation. Knowledge of their mechanism of action may suggest novel approaches to the cancer problem and, as in the case of vitamins A and D, it may be possible to find analogues with more potent anti-cancer activity.

Role of dietary lysine, methionine, and arginine in the regulation of hypercholesterolemia in rabbits.

These experiments were conducted to see whether the hypercholesterolemia produced by a diet enriched in lysine (Lys) and methionine (Met) can be reproduced by feeding these amino acids separately, and whether dietary arginine (Arg) counteracts their hypercholesterolemic effects. Another aim was to investigate the mechanisms involved in modulations of serum cholesterol levels by these amino acids. The results of this study, which were in agreement with the results of earlier experiments in our laboratory, showed that feeding a low-fat, cholesterol-free, semipurified amino acid diet enriched with Lys + Met to rabbits caused a marked increase in serum total and low density lipoprotein cholesterol and apolipoprotein B levels, whereas a similar diet enriched in essential ketogenic amino acids (EketoAA) resulted in a more moderate increase in these parameters. Supplementing the diet with either Lys or Met alone was also less effective in inducing hypercholesterolemia than increasing levels of both amino acids. Dietary Arg partially counteracted the hypercholesterolemic effect of Lys + Met but not that of the EketoAA or of Lys and Met fed separately. The growth performance of rabbits fed the Lys + Met diet was inferior to that of those fed the other diets. Liver total phospholipid levels and the ratio of phosphatidylcholine to phosphatidylethanolamine were higher in rabbits fed the Lys + Met-enriched diet than in those animals fed a diet in which Arg was supplemented. In conclusion, our results indicate that high levels of both Lys and Met are needed to cause a maximum elevation of serum cholesterol and that the moderately antihypercholesterolemic effect of Arg is seen only when both amino acids are supplemented. They also suggest that these essential amino acids may affect cholesterol metabolism partly through alteration of liver phospholipids.

Obesity as a risk factor for certain types of cancer.

In conclusion, obesity has been associated with increased risk for a number of different types of cancer. The evidence has been most consistent for endometrial cancer, breast cancer in postmenopausal women, and renal cell cancer. More variable results have been reported for colorectal, prostate and pancreatic cancer. Possible mechanisms by which obesity may influence cancer risk include alteration in hormonal patterns, including sex hormones and insulin, and factors such as the distribution of body fat and changes in adiposity at different ages. The increasing prevalence of obesity in many parts of the world emphasizes the importance of learning more about the relationship between obesity and cancer and the mechanisms involved in their interaction.

Inhibition of mammary cancer by citrus flavonoids.

Double strength orange juice given to the rats in place of drinking water inhibited mammary tumorigenesis induced in female Sprague-Dawley rats by DMBA more effectively than double strength grapefruit juice. This may mean that hesperetin retains its effectiveness in vivo better than naringenin, since the flavonoids are present in the juices at similar levels. It is also possible that orange juice contains other compounds that have anti-cancer activity and that may act synergistically with hesperetin. Citrus flavonoids are effective inhibitors of both estrogen receptor-negative MDA-MB-435 and estrogen receptor-positive MCF-7 human breast cancer cell in vitro. Furthermore, 1:1 combinations of flavonoids with tocotrienols and/or tamoxifen inhibit proliferation of the cells more effectively than the individual compounds. This synergism may be due to the fact that the compounds are exerting their inhibitory effects by different mechanisms.

Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors.

Previous results suggested that changes in the activity of nitric oxide (NO) can influence metabolism of apo B-containing lipoproteins. Therefore, we studied effects of exogenous NO donors and physiological NO precursors on metabolism of these lipoproteins. In rabbits, addition of 0.03% sodium nitroprusside (NaNP) to a semipurified, cholesterol-free, casein diet counteracted the elevation of LDL cholesterol induced by this diet but did not alter liver lipids after 4 weeks of feeding. In HepG2 cells, addition of nontoxic concentrations of another NO donor, S-nitroso-N-acetylpenicillamine (SNAP) to culture medium caused a dose-dependent reduction of medium apo B after 24 h. At the concentration 0.5 mM, SNAP significantly decreased medium apo B by 50% without altering total synthesis and secretion of proteins and without altering rates of cellular sterol synthesis. In cells incubated with L-arginine, reduction of medium apo B was not associated with increased NO production whereas in those exposed to N-OH-Arg medium apo B levels were not altered. We concluded that synthetic NO donors can reduce hypercholesterolemia by affecting apo B metabolism directly in the liver, via the sterol-independent mechanism.

Mouse running activity is lowered by Brucella abortus treatment: a potential model to study chronic fatigue.

Chronic fatigue syndrome, which can occur after acute infection and last for years, is characterized by severe and persistent fatigue. Others have reported decreases in mouse running activity following infection and have suggested this may provide an animal model for studying chronic fatigue. Voluntary running is a highly motivated activity in mice, which will often run 5-7 mi/day in our laboratory. Following 2 weeks of acclimation to running wheels with food and water available ad lib, female BALB/c mice received 0.2-mL tail vein injections of killed Brucella abortus (BA) or saline vehicle. Subsequently the effects on voluntary running and grooming behavior were determined. Injection of BA caused an immediate large decrease in running and a lack of grooming. Vehicle injections produced no changes in behavior. After the first several days of reduced running behavior, levels of running and grooming slowly returned back to normal over the next 2-4 weeks, with substantial individual differences in the rate of recovery. The pattern of running during recovery was intriguing in that BA mice first ran at normal levels just after the lights went out, but they stopped after only 1-2 h. As recovery proceeded, they gradually increased the duration of the running bout during the night. Because this model uses voluntary exertion and the ability to run for longer periods of time characterizes recovery, the model may be a good one for studying the biologic underpinnings of chronic fatigue.

Cross training: indices of training stress and performance.

Twenty well-trained runners (VO2max 4.6+/-0.5 L x min[-1]) were age and ability matched and assigned to either a cross training (CT) or run only group (RT). All subjects maintained normal running distance and intensity for 6 wk and reported for three additional training sessions per week. These workouts were performed outdoors on a 400-m track or measured road course (RT) or on a bicycle ergometer (CT). The sessions were as follows: (work x rest(-1) ratio = 1): 5 x 5 min at >95% VO2max/peak (Monday), 50-60 min at 70% VO2max/peak (Wednesday), and 3 x 2.5 min at >105% VO2max/peak, plus 6 x 1.25 min at >115% VO2max/peak (Friday). Subjects were tested before (PRE), after 3 wk (MID), and after 6 wk (POST) of intensified training. Blood samples were obtained from RT, CT, and ten controls (CON) at each time point (0600 h). Runners also completed a 10-min submaximal run at the same absolute intensity (velocity to elicit 75% of initial V02max) during which heart rate, RPE, and VO2 were measured. Each runner then completed a simulated 5-km race (time trial) on a treadmill. Total testosterone (TT), free testosterone (FT), cortisol (C), and creatine kinase activity (CK) were determined. Running economy was similar between RT and CT; however, RPE decreased significantly at MID and POST compared with that at PRE (P < 0.05; time effect). There were no significant differences among groups for TT, FT, or CK, but C was significantly lower in CON than in RT and CT. Performance was significantly faster (P < 0.05; time effect) in the 5-km race at MID (1076.1+/-81.4 s) and POST (1068.6+/-83.9) compared with PRE (1096.6+/-79.5) but was not different between CT and RT. In conclusion, RT and CT responded similarly to 6 wk of increased training, and both groups improved 5-km performance to a similar extent.

Effects of substituting dietary soybean protein and oil for milk protein and fat in subjects with hypercholesterolemia.

OBJECTIVES: To determine whether, in individuals with hypercholesterolemia, substituting dietary soybean products for cows' milk products improves the plasma lipid profile and whether any change in the profile is due partially to soy oil. DESIGN: Randomized 3-treatment crossover trial. SETTING: Family practice clinics and an outpatient clinic in London, Ont. PARTICIPANTS: Seventeen healthy men and 17 healthy women with elevated plasma levels of total and low-density-lipoprotein (LDL) cholesterol and with normal plasma levels of triglycerides. INTERVENTIONS: Participants incorporated into their normal diet either 2% cows' milk products, soybean products or a combination of skim milk products and soy oil, each over period of 4 weeks, with 22-week wash-out periods. Plasma lipid profile, blood pressure and body weight were assessed after each dietary and wash-out period. OUTCOME MEASURES: Plasma levels of total and lipoprotein cholesterol, plasma levels of triglycerides, apolipoprotein B and A1 levels, blood pressure and plasma lipid peroxidation. RESULTS: The change in diet had no effect on body mass index, levels of apolipoproteins B and A1 and most plasma lipids. During the soybean period, the subjects' mean level of high-density-lipoprotein (HDL) cholesterol increased 9% (p < 0.04) and their mean LDL/HDL cholesterol ratio decreased 14% (p < 0.007). These effects were less pronounced during the skim milk/soy oil period. In the 24 subjects with the highest initial LDL cholesterol level and LDL/HDL cholesterol ratio, the mean LDL cholesterol level decreased 11% after the soybean period. In all subjects, changes in the LDL/HDL cholesterol ratio induced by a soybean diet were negatively correlated with the initial LDL/HDL cholesterol ratio and positively correlated with the initial HDL cholesterol level. CONCLUSIONS: In people with hypercholesterolemia, the plasma lipid profile improved after treatment with a soybean-product diet, and this improvement was partially due to soy oil. The degree of responsiveness was associated with initial risk factors for coronary artery disease.

Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination.

Tocotrienols are a form of vitamin E, having an unsaturated isoprenoid side-chain rather than the saturated side-chain of tocopherols. The tocotrienol-rich fraction (TRF) from palm oil contains alpha-tocopherol and a mixture of alpha-, gamma- and delta-tocotrienols. Earlier studies have shown that tocotrienols display anticancer activity. We previously reported that TRF, alpha-, gamma- and delta-tocotrienols inhibited proliferation of estrogen receptor-negative MDA-MB-435 human breast cancer cells with 50% inhibitory concentrations (IC50) of 180, 90, 30 and 90 microg/mL, respectively, whereas alpha-tocopherol had no effect at concentrations up to 500 microg/mL. Further experiments with estrogen receptor-positive MCF-7 cells showed that tocotrienols also inhibited their proliferation, as measured by [3H] thymidine incorporation. The IC50s for TRF, alpha-tocopherol, alpha-, gamma- and delta-tocotrienols were 4, 125, 6, 2 and 2 microg/mL, respectively. Tamoxifen, a widely used synthetic antiestrogen inhibits the growth of MCF-7 cells with an IC50 of 0.04 microg/mL. We tested 1:1 combinations of TRF, alpha-tocopherol and the individual tocotrienols with tamoxifen in both cell lines. In the MDA-MB-435 cells, all of the combinations were found to be synergistic. In the MCF-7 cells, only 1:1 combinations of gamma- or delta-tocotrienol with tamoxifen showed a synergistic inhibitory effect on the proliferative rate and growth of the cells. The inhibition by tocotrienols was not overcome by addition of excess estradiol to the medium. These results suggest that tocotrienols are effective inhibitors of both estrogen receptor-negative and -positive cells and that combinations with tamoxifen should be considered as a possible improvement in breast cancer therapy.

The role of dietary fat in breast cancer.

Studies on experimental animals have generally supported a role for dietary fat in mammary cancer, but epidemiological studies have given conflicting results. The effects of dietary fat in animals may be due to its high energy density or to other specific effects, and may have relevance for human breast cancer.

Inhibition of proliferation of estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen.

The flavonoids are a group of naturally-occurring, low molecular weight compounds that are widespread in plants. Representatives of several different classes of flavonoids were tested for their effects on the proliferation of an estrogen receptor-positive human breast cancer cell line, MCF-7. The IC50S (concentration at which cell proliferation was inhibited by 50%), based on [3H]thymidine incorporation, ranged from 4.2 to 18.0 micrograms/mL. The cells were viable at these concentrations. The possibility that flavonoids may block cell proliferation by binding to the estrogen receptor was explored. The cells were depleted of endogenous steroids and incubated with individual flavonoids at their IC50 concentration. Half of the cells were exposed to an excess concentration of 17 beta-estradiol to see if this affected antiproliferation by the flavonoids. Of the flavonoids tested, only the inhibition of cell proliferation by genistein was reversed with the addition of excess, competing estrogen. Baicalein, galangin, hesperetin, naringenin and quercetin apparently exert their antiproliferative activity via some other mechanism.

Palm oil tocotrienols and plant flavonoids act synergistically with each other and with Tamoxifen in inhibiting proliferation and growth of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells in culture.

Palm oil, unlike many other dietary oils, does not increase the yield of chemically-induced mammary tumors in rats when fed at high levels in the diet. This difference appears to be due to the vitamin E fraction of palm oil, which is rich in tocotrienols, since palm oil stripped of this fraction does increase tumor yields. Experiments in our laboratory have shown that tocotrienols inhibit proliferation and growth of both MDA-MB-435 and MCF-7 cells in culture much more effectively than a-tocopherol. In addition, it was found that combinations of tocotrienols with Tamoxifen, a drug widely used for treatment of breast cancer, inhibit these cells more effectively than either tocotrienols or Tamoxifen alone. The present studies have now shown synergistic effects between tocotrienols and a number of other flavonoids from various plant sources, including citrus fruits, in the inhibition of both MDA-MB-435 and MCF-7 cells (IC50s 0.05-25 and 0.02-5 µg/mL respectively). In the MCF-7 cells, 1:1:1 combinations of tocotrienols, flavonoids and Tamoxifen were even more effective, with the best combination being d-tocotrienol, hesperetin and Tamoxifen (IC50 0.0005 µg/mL). These results suggest that diets containing palm oil may reduce the risk of breast cancer, particularly when eaten with other plant foods containing flavonoids, and may also enhance the effectiveness of Tamoxifen for treatment of breast cancer.

Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices.

Two citrus flavonoids, hesperetin and naringenin, found in oranges and grapefruit, respectively, and four noncitrus flavonoids, baicalein, galangin, genistein, and quercetin, were tested singly and in one-to-one combinations for their effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50), based on incorporation of [3H]thymidine, varied from 5.9 to 140 micrograms/ml for the single flavonoids, with the most potent being baicalein. IC50 values for the one-to-one combinations ranged from 4.7 micrograms/ml (quercetin + hesperetin, quercetin + naringenin) to 22.5 micrograms/ml (naringenin + hesperetin). All the flavonoids showed low cytotoxicity (> 500 micrograms/ml for 50% cell death). Naringenin is present in grapefruit mainly as its glycosylated form, naringin. These compounds, as well as grapefruit and orange juice concentrates, were tested for their ability to inhibit development of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Two experiments were conducted in which groups of 21 rats were fed a semipurified diet containing 5% corn oil and were given a 5-mg dose of DMBA intragastrically at approximately 50 days of age while in diestrus. One week later, individual groups were given double-strength grapefruit juice or orange juice or fed naringin or naringenin at levels comparable to that provided by the grapefruit juice; in the second experiment, the rats were fed a semipurified diet containing 20% corn oil at that time. As expected, rats fed the high-fat diet developed more tumors than rats fed the low-fat diet, but in both experiments tumor development was delayed in the groups given orange juice or fed the naringin-supplemented diet compared with the other three groups. Although tumor incidence and tumor burden (grams of tumor/rat) were somewhat variable in the different groups, rats given orange juice had a smaller tumor burden than controls, although they grew better than any of the other groups. These experiments provide evidence of anticancer properties of orange juice and indicate that citrus flavonoids are effective inhibitors of human breast cancer cell proliferation in vitro, especially when paired with quercetin, which is widely distributed in other foods.