Influence of Obesity on Breast Density Reduction by Omega-3 Fatty Acids: Evidence from a Randomized Clinical Trial.

Preclinical data indicate that omega-3 fatty acids (n-3FA) potentiate the chemopreventive effect of the antiestrogen (AE) tamoxifen against mammary carcinogenesis. The role of n-3FA in breast cancer prevention in humans is controversial. Preclinical and epidemiologic data suggest that n-3FA may be preferentially protective in obese subjects. To directly test the protective effect of n-3FA against breast cancer, we conducted a 2-year, open-label randomized clinical trial in 266 healthy postmenopausal women (50% normal weight, 30% overweight, 20% obese) with high breast density (BD; ≥25%) detected on their routine screening mammograms. Eligible women were randomized to one of the following five groups (i) no treatment, control; (ii) raloxifene 60 mg; (iii) raloxifene 30 mg; (iv) n-3FA lovaza 4 g; and (v) lovaza 4 g plus raloxifene 30 mg. The 2-year change in BD, a validated biomarker of breast cancer risk, was the primary endpoint of the study. In subset analysis, we tested the prespecified hypothesis that body mass index (BMI) influences the relationship between plasma n-3FA on BD. While none of the interventions affected BD in the intention-to-treat analysis, increase in plasma DHA was associated with a decrease in absolute breast density but only in participants with BMI >29. Our results suggest that obese women may preferentially experience breast cancer risk reduction from n-3FA administration.

Randomized controlled trial of oral glutathione supplementation on body stores of glutathione.

PURPOSE: Glutathione (GSH), the most abundant endogenous antioxidant, is a critical regulator of oxidative stress and immune function. While oral GSH has been shown to be bioavailable in laboratory animal models, its efficacy in humans has not been established. Our objective was to determine the long-term effectiveness of oral GSH supplementation on body stores of GSH in healthy adults. METHODS: A 6-month randomized, double-blinded, placebo-controlled trial of oral GSH (250 or 1,000 mg/day) on GSH levels in blood, erythrocytes, plasma, lymphocytes and exfoliated buccal mucosal cells was conducted in 54 non-smoking adults. Secondary outcomes on a subset of subjects included a battery of immune markers. RESULTS: GSH levels in blood increased after 1, 3 and 6 months versus baseline at both doses. At 6 months, mean GSH levels increased 30-35 % in erythrocytes, plasma and lymphocytes and 260 % in buccal cells in the high-dose group (P < 0.05). GSH levels increased 17 and 29 % in blood and erythrocytes, respectively, in the low-dose group (P < 0.05). In most cases, the increases were dose and time dependent, and levels returned to baseline after a 1-month washout period. A reduction in oxidative stress in both GSH dose groups was indicated by decreases in the oxidized to reduced glutathione ratio in whole blood after 6 months. Natural killer cytotoxicity increased >twofold in the high-dose group versus placebo (P < 0.05) at 3 months. CONCLUSIONS: These findings show, for the first time, that daily consumption of GSH supplements was effective at increasing body compartment stores of GSH.

Comparative effects of two different forms of selenium on oxidative stress biomarkers in healthy men: a randomized clinical trial.

Epidemiologic and laboratory studies indicate that dietary selenium protects against prostate cancer. Results from clinical trials suggest that selenium-enriched yeast (SY) but not selenomethionine (SeMet) may be effective at reducing prostate cancer risk. Our objectives were to directly compare for the first time the effects of SeMet and SY on prostate cancer relevant biomarkers in men. We performed a randomized double blind, placebo-controlled trial of SY (200 or 285 µg/day) and SeMet (200 µg/day) administered for 9 months in 69 healthy men. Primary endpoints included blood levels of selenium-containing compounds and oxidative stress biomarkers [urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin-F2α (8-iso-PGF2α) and blood glutathione (GSH)]. Secondary endpoints included plasma glucose and PSA levels. Compliance was high in all groups (>95%). Plasma selenium levels were increased 93%, 54%, and 86% after 9 months in SeMet and low- and high-dose SY groups, respectively, and returned to baseline levels after a 3-month washout (P < 0.05). Levels of 8-OHdG and 8-iso-PGF2α were decreased 34% and 28%, respectively, after 9 months in the high-dose SY group (P < 0.05). These decreases were greatest in individuals with low baseline plasma levels of selenium (<127 ng/mL). No changes in serum PSA or blood glucose and GSH were observed. Overall, we showed for the first time, reductions in biomarkers of oxidative stress following supplementation with SY but not SeMet in healthy men. These findings suggest that selenium-containing compounds other than SeMet may account for the decrease in oxidative stress.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis.

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

Age related changes in selenium and glutathione levels in different lobes of the rat prostate.

Aging represents a major risk factor for prostate cancer; however, mechanisms responsible for this relationship remain unclear. Preclinical and some clinical investigations support the protective role of selenium against prostate cancer possibly through the reduction of oxidative stress. While increased levels of oxidative stress together with decreases in selenium and the major cellular antioxidant glutathione (GSH) are common in tissues of old animals, there is little data available on these parameters in the prostate. In the present study we have compared the levels of selenium, GSH and protein-bound GSH (GSSP) in blood and prostate tissues in young (4-month), mature (12-month), old (18 month), and very old (24 month) male F344 rats. Each prostate lobe (dorsolateral, DL; anterior, AL; ventral, VL) was analyzed separately based upon their differing potential for prostate cancer development. At all ages, selenium levels were lowest in DL

3,5,5-trimethyl-hexanoyl-ferrocene diet protects mice from moderate transient acetaminophen-induced hepatotoxicity.

Acetaminophen (APAP) overdose is the most frequent cause of adult acute liver failure. Susceptibility or resistance to APAP toxicity is most likely accounted for by the interplay of several factors. One factor important in multiple different chronic liver diseases that may play a role in APAP toxicity is elevated hepatic iron. Hereditary hemochromatosis is traditionally associated with hepatic iron overload. However, varying degrees of elevated hepatic iron stores observed in chronic hepatitis C and B, alcoholic liver disease and nonalcoholic fatty liver disease also have clinical relevance. We employed an animal model in which mice are fed a 3,5,5-trimethyl-hexanoyl-ferrocene (TMHF)-supplemented diet to evaluate the effect of elevated hepatic iron on APAP hepatotoxicity. Three hundred milligrams per kilogram APAP was chosen because this dosage induces hepatotoxicity but is not lethal. Since both excess iron and APAP induce oxidative stress and mitochondrial dysfunction, we hypothesized that the TMHF diet would enhance APAP hepatotoxicity. The results were the opposite. Centrilobular vacuolation/necrosis, APAP adducts, nitrotyrosine adducts, and a spike in serum alanine aminotransferase, which were observed in control mice treated with APAP, were not observed in TMHF-fed mice treated with APAP. Further analysis showed that the levels of CYP2E1 and CYP1A2 were not significantly different in TMHF-treated compared with control mice. However, the magnitude of depletion of glutathione following APAP treatment was considerably less in TMHF-treated mice than in mice fed a control diet. We conclude that a TMHF diet protects mice from moderate transient APAP-induced hepatotoxicity prior to the formation of APAP adducts, and one contributing mechanism is reduction in glutathione depletion.

Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.

Chemoprevention of breast cancer by fish oil in preclinical models: trials and tribulations.

Despite the perception that omega-3 fatty acids (n-3 FA) protect against breast cancer, epidemiologic studies have yielded inconsistent results. Although preclinical data have been, in general, more supportive of a protective effect of n-3 FA on breast cancer, inconsistencies still remain, which preclude definite conclusions or in-depth mechanistic investigations despite 30 years of research in this area. In this review, we discuss key variables that may account for inconsistencies of results across preclinical studies and provide recommendations for future experiments testing the chemopreventive effect of n-3 FAs in breast cancer, as part of a multiagent approach under rigorously controlled conditions.

Mammary gland density predicts the cancer inhibitory activity of the N-3 to N-6 ratio of dietary fat.

This study investigated the effect of a broad range of dietary ratios of n-3:n-6 fatty acids on mammary gland density and mammary cancer risk. Cancer was induced in female rats by N-methyl-N-nitrosourea. Purified diet that provided 30% of dietary kilocalories from fat was formulated to contain ratios of n-3:n-6 fatty acids from 25:1 to 1:25. Mammary gland density was determined by digital analysis, fatty acids by gas chromatography/flame ionization detection, and other plasma analytes via ELISA. Mammary gland density was reduced dose dependently at n-3:n-6 ratios from 1:1 to 25:1 (r = -0.477, P = 0.038), with a 20.3% decrease of mammary gland density between n-3:n-6 of 1:1 versus 25:1, P < 0.001. Mammary carcinogenesis was inhibited in the absence or presence of tamoxifen (1 mg/kg diet) in a manner predicted by mammary gland density. Plasma n-3 fatty acid concentrations failed to increase above an n-3:n-6 ratio of 5:1, and changes in specific plasma n-3 or n-6 fatty acids were not predictive of mammary gland density or cancer inhibitory activity. A strong reciprocal effect of the n-3:n-6 ratio on plasma leptin (decreased, P = 0.005) and adiponectin (increased, P < 0.001) was observed indicating adipose tissue function was modulated. However, neither cytokine was predictive of mammary gland density. Plasma insulin-like growth factor I (IGF-I) decreased with increasing dietary n-3:n-6 ratio (P = 0.004) and was predictive of the changes in mammary gland density (r = 0.362, P < 0.005). These findings indicate that (i) mammary gland density predicted the carcinogenic response, (ii) the n-3:n-6 ratio exerts effects in the presence or absence of hormonal regulation of carcinogenesis, and (iii) signaling pathways regulated by IGF-I are potential targets for further mechanistic investigation.

Association of selenium status and blood glutathione concentrations in blacks and whites.

Selenium deficiency has been linked with increased cancer risk and, in some studies, selenium supplementation was protective against certain cancers. Previous studies have suggested that selenium chemoprevention may involve reduced oxidative stress through enhanced glutathione (GSH). Our objectives were to examine the relationships between selenium and GSH in the blood and the modifying effects of race and sex in free-living adults and individuals supplemented with selenium. Plasma selenium concentrations and free and bound GSH concentrations and γ-glutamyl cysteine ligase (GCL) activity in the blood were measured in 336 healthy adults (161 Blacks, 175 Whites). Plasma selenium and blood GSH were also measured in 36 healthy men from our previously conducted placebo-controlled trial of selenium-enriched yeast (247 µg/day for 9 mo). In free-living adults, selenium concentrations were associated with increased blood GSH concentration and GCL activity (P < 0.05). Further, selenium was significantly higher in Whites than in Blacks (P < 0.01). After 9 mo of supplementation, plasma selenium increased 114% in Whites and 50% in Blacks (P < 0.05), and blood GSH increased 35% in Whites (P < 0.05) but was unchanged in Blacks. These results indicate a direct association between selenium and GSH in the blood of both free-living and selenium-supplemented individuals, with race being an important modifying factor.

Selenium-responsive proteins in the sera of selenium-enriched yeast-supplemented healthy African American and Caucasian men.

BACKGROUND: Studies have shown that supplementation of adult men with selenium-enriched yeast (SY) was protective against prostate cancer (PCa) and also reduced oxidative stress and levels of prostate-specific antigen. Here, we determined the effect of SY supplementation on global serum protein expression in healthy men to provide new insights into the mechanism of selenium chemoprevention; such proteins may also serve as biomarkers of disease progression. METHODS: Serum samples from 36 adult men were obtained from our previous SY clinical trial, 9 months after supplementation with either SY (247 microg/d; n = 17) or placebo (nonenriched yeast; n = 19). RESULTS: Proteomic profiling using two-dimensional difference in gel electrophoresis followed by liquid chromatography-tandem mass spectrometry revealed a total of 1,496 candidate proteins, of which, 11 were differentially expressed in the SY group as compared with placebo. Eight proteins were upregulated [clusterin isoform 1 (CLU), transthyretin, alpha-1B-glycoprotein, transferrin, complement component 4B proprotein, isocitrate dehydrogenase, haptoglobin, and keratin 1] and three proteins were downregulated [alpha-1 antitrypsin (AAT), angiotensin precursor, and albumin precursor] by SY. All of the identified proteins were redox-sensitive or involved in the regulation of redox status. Because both AAT and CLU have been previously linked to PCa development, their identities were confirmed by two-dimensional Western blot analysis. CONCLUSIONS: We identified AAT and CLU as potential candidate proteins involved in the mechanism of PCa prevention by SY. Collectively, proteins identified in this study might serve as potential new biomarkers for monitoring and comparing responses to selenium-based chemopreventive agents. IMPACT: Proteomic analysis of serum might be useful for the early detection and monitoring efficacy of chemopreventive agents.

The impact of fish oil on the chemopreventive efficacy of tamoxifen against development of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies.

The organoselenium compound 1,4-phenylenebis(methylene)selenocyanate inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorgenesis and enhances glutathione-related antioxidant levels in A/J mouse lung.

Selenium, in the form of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) but not Se-enriched yeast (Se-yeast), was highly effective at inhibiting lung tumors induced by the tobacco specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and at reducing NNK-induced DNA methylation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the lung. Our goal was to determine if p-XSC but not Se-yeast is effective at inducing levels of glutathione (GSH)-related antioxidants and reducing markers of GSH oxidation in the NNK-induced lung tumor model. In the first bioassay, 6-week-old mice were fed either control or experimental diets (containing 10 ppm as selenium from p-XSC or Se-yeast) and, beginning at 8 weeks of age, received NNK (3 micromol) by gavage once weekly for 8 weeks. After 18 weeks, p-XSC significantly reduced NNK-induced tumor burden by 74% (10.4 +/- 6.0 versus 2.7 +/- 1.5 tumors/mouse, P < 0.001) and tumor incidence from 96% to 68% (P < 0.01), whereas, Se-yeast had no effect. Lung GSH levels were unchanged by either NNK or Se-yeast, but were increased 70% in mice treated with both NNK and p-XSC (P < 0.01) and 41% in mice treated with p-XSC alone. In the second bioassay, the time course of effects of p-XSC was examined. As early as one week after initiation of p-XSC feeding lung and blood selenium levels were increased nearly six- and two-fold, respectively. Increases of 120% for GSH and 65% for Cys were observed in p-XSC groups compared to controls within one week after initiation of p-XSC feeding (P < 0.01). The levels of protein-bound:free GSH ratios and Cys ratios were significantly decreased in p-XSC-treated mice, regardless of NNK status, suggesting a decrease in the levels of oxidative stress. Altogether, these results indicate that p-XSC is a potent inducer of GSH and related thiol antioxidants in the lung leading to decreased levels of oxidative stress and suggest that p-XSC inhibits tumor formation, in part, by protecting against oxidative damage.

Influence of selenium-enriched yeast supplementation on biomarkers of oxidative damage and hormone status in healthy adult males: a clinical pilot study.

The mechanisms responsible for the protective role of selenium against the development of prostate cancer remain to be determined (L. C. Clark et al., J. Am. Med. Assoc., 276: 1957-1963, 1996). In the present study, we tested the hypothesis that selenium supplementation reduces oxidative stress. A secondary aim was to determine whether selenium-induced changes in testosterone (T) metabolism may also be involved. To this end, we conducted a double-blind, randomized, placebo-controlled trial of 247 micro g selenium/day administered p.o. in the form of Se-enriched yeast. Study subjects were 36 healthy adult males, 11 blacks and 25 whites, 19-43 years of age. Supplementation occurred over the first 9 months, after which all subjects were placed on placebo for an additional 3 months. Blood and urine were collected at baseline and after 3, 9, and 12 months. In the selenium group, plasma selenium levels were 2-fold higher than baseline values after 3 and 9 months and returned to 136% of baseline after 12 months (P < 0.0001), whereas in the placebo group, levels were unchanged. A 32% increase in blood glutathione (GSH) levels was observed after 9 months in the selenium group only (P < 0.05). This change coincided with a 26% decrease in protein-bound GSH (bGSH) and a 44% decrease in bGSH:GSH ratios (P < 0.05). The changes in GSH and bGSH were highly correlated with changes in plasma selenium concentrations and may reflect a decrease in oxidative stress. No changes were observed in either group for plasma T, dihydrotestosterone (DHT) or DHT:T ratios, suggesting that selenium had no effect on the alpha-reductase involved in the conversion of T to DHT. A small but significant decrease in prostate-specific antigen levels was observed after 3 and 9 months (P < 0.001), and this difference disappeared after 12 months. Future trials will test the above hypothesis in prostate cancer patients and in subjects at high risk for prostate cancer.