Deleterious effects of high concentrations of (-)-epigallocatechin-3-gallate and atorvastatin in mice with colon inflammation.

Epigallocatechin-3-gallate (EGCG), atorvastatin (ATST), and their combination have been previously shown to inhibit colon carcinogenesis in animal models. We further investigated their inhibitory activities in azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated Balb/cJ mice and CD-1 mice in 2 slightly different models. The mice were maintained on the AIN93M diet, or a similar diet containing 0.03%, 0.1%, or 0.3% EGCG; 60-ppm ATST; or a combination of 0.1% EGCG and 60-ppm ATST. Unexpectedly, no significant inhibitory activity was observed, and some of the treatment groups resulted in higher tumor multiplicity. To study the effects of EGCG on colon inflammation, CD-1 or C57BL/6 mice were treated with 1.5% DSS for 7 days and sacrificed 3 days later. DSS induced rectal bleeding and colon shortening; treatment with 0.5% EGCG exacerbated the bleeding and decreased mouse body weight. Dietary 0.5% EGCG also increased serum levels of leukotriene B4 and prostaglandin E2. These results suggest that, in mice bearing colon inflammation, high concentrations of EGCG and ATST enhance colon bleeding and may promote colon carcinogenesis.

Rapid induction of colon carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced during the cooking of meats and fish, is suspected to be a human carcinogen. Metabolic activation of PhIP is primarily mediated by the enzyme cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2 differs considerably between humans and rodents, with more N(2)-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Transgenic CYP1A-humanized mice (hCYP1A-mice), which have the human CYP1A1 and CYP1A2 genes but lack the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent opportunity to develop a relevant model to study dietary-induced colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral gavage, followed by 1.5% dextran sodium sulfate (DSS) in the drinking water for 7 days, was found to be an effective combination to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplicity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was 3.90 ± 0.61 with 80-95% of the tumors being adenocarcinomas. No tumors were found in the similarly treated wild-type mice. Western blots revealed overexpression of ß-catenin, c-Myc, cyclin D1, inducible nitric oxide synthase and cyclooxygenase-2 in colon tumor samples. Strong nuclear localization of ß-catenin was observed in tumors. These results illustrate that PhIP and DSS combination produces rapid colon carcinogenesis in hCYP1A-mice and this is an effective model to mimic human colon carcinogenesis.

The Effects of Green Tea on Diabetes and Gut Microbiome in db/db Mice: Studies with Tea Extracts vs. Tea Powder.

Green tea extracts and tea catechins have been shown to prevent or alleviate diabetes. The present study tests the hypothesis that green tea leaves in powder form (GTP), which also contain fiber and other water non-extractable materials, are more effective than the corresponding green tea extracts (GTE) in impeding the development of diabetes in db/db mice. Female db/db mice were treated with a diet containing 1% of GTE, 2% of GTE, 2% of GTP (with the same catechin content as 1% GTE) or 1% GTP. The 1% GTE group had lower food intake, water consumption, body weight and fasting blood glucose levels than the control group, while 2% GTP did not have any significant effect. Dietary 1% GTE also preserved ß-cell insulin secretion. However, 1% GTP increased food intake, water consumption and blood glucose levels. Microbiome analysis with 16S rRNA gene V4 sequencing showed that the gut microbiota was modified by GTE and GTP, and a few bacterial guilds were associated with blood glucose levels. In the Random Forest regression model, the leading predictor of metabolic outcome was food consumption, followed by changes in some bacterial guilds. The results illustrate the importance of food consumption and gut microbiota in affecting the progression of diabetes.

Protective effects of α-galacto-oligosaccharides against a high-fat/western-style diet-induced metabolic abnormalities in mice.

In this study, we investigated the effects of a newly synthesized α-galacto-oligosaccharide mixture (α-GOSg), 0.5% in drinking water, on high-fat/western-style diet (HFWD)-induced metabolic abnormality in mice in a study of 13 weeks. Raffinose family oligosaccharides (RFOs) were included as a comparison. Mice treated with α-GOSg had significantly lower body weight and body fat (p < 0.05), while RFOs were less effective. Both α-GOSg and RFOs significantly reduced serum levels of total cholesterol and low-density lipoprotein cholesterol, alanine aminotransferase and liver lipids. However, only α-GOSg significantly decreased the histopathological score for liver steatosis and downregulated hepatic fatty acid synthesis gene acetyl CoA carboxylase-α. α-GOSg also significantly reduced the content of bile acids in the small intestine and significantly increased the abundance of gut Bifidobacterium and decreased the abundance of Clostridium leptum. These actions are proposed to be key mechanisms contributing to the beneficial health effects of α-GOSg.

Effects of antibiotics on degradation and bioavailability of different vitamin E forms in mice.

Tocopherols (T) and tocotrienols (T3), all existing in α, ß, γ, and δ-forms, are the eight forms of vitamin E (VE). In this study, we investigated the effects of gut microbiota on the degradation and tissue levels of different VE forms by treating mice with antibiotics in drinking water for 12 days. The mice also received an intragastric (i.g.) dose of VE mixture (mVE; α-T, γ-T, δ-T, γ-T3, and δ-T3, each at a dose of 75 mg/kg) every morning. Antibiotic treatment significantly increased the blood levels of all VE forms in mice that received an i.g. dose of mVE in the morning, 3 h before sacrifice. Without this morning dose, the blood levels of α-T were at the normal physiological levels, but those of the other VE forms were much lower; and the levels of all VE forms were not significantly affected by antibiotics. The liver levels of these VE forms were generally higher and followed the same pattern as the serum. On the contrary, the levels of most side-chain degradation metabolites of VE forms in the serum, liver, kidney, urine, and fecal samples were significantly decreased by antibiotics. The increased bioavailability of VE by antibiotics is probably due to increased absorption of VE or its decreased degradation by gut microbes. The results demonstrate the important roles of gut microbiota in the degradation of VE and in decreasing the bioavailabilities of VE forms. © 2019 BioFactors, 45(3):450-462, 2019.

Green Tea Polyphenols Modify the Gut Microbiome in db/db Mice as Co-Abundance Groups Correlating with the Blood Glucose Lowering Effect.

SCOPE: The effects of green tea polyphenols, Polyphenon E (PPE), and black tea polyphenols, theaflavins (TFs), on gut microbiota and development of diabetes in db/db mice are investigated and compared. METHODS AND RESULTS: Supplementation of PPE (0.1%) in the diet of female db/db mice for 7 weeks decreases fasting blood glucose levels and mesenteric fat while increasing the serum level of insulin, possibly through protection against ß-cell damage. However, TFs are less or not effective. Microbiome analysis through 16S rRNA gene sequencing shows that PPE and TFs treatments significantly alter the bacterial community structure in the cecum and colon, but not in the ileum. The key bacterial phylotypes responding to the treatments are then clustered into 11 co-abundance groups (CAGs). CAGs 6 and 7, significantly increased by PPE but not by TFs, are negatively associated with blood glucose levels. The operational taxonomic units in these CAGs are from two different phyla, Firmicutes and Bacteroidetes. CAG 10, decreased by PPE and TFs, is positively associated with blood glucose levels. CONCLUSION: Gut microbiota respond to tea polyphenol treatments as CAGs instead of taxa. Some of the CAGs associated with the blood glucose lowering effect are enriched by PPE, but not TFs.

Effects of gut microbiota and time of treatment on tissue levels of green tea polyphenols in mice.

The previous studies have shown that tea polyphenols are metabolized by gut microbiota. This study investigated the effect of gut microbiota on the bioavailability, tissue levels, and degradation of tea polyphenols. Mice were treated with antibiotics (ampicillin/sulfamethoxazole/trimethoprim) in drinking water and the control mice received water for 11 days, and they were given an AIN93M diet enriched with 0.32% of Polyphenon E. The levels of catechins and their metabolites (if present) in the serum, liver, urine, and fecal samples were determined by high-performance liquid chromatography. The results showed that treatment with antibiotics significantly increased the levels of the major polyphenol, (-)-epigallocatechin-3-gallate (EGCG), in serum and liver samples. Antibiotics also raised the levels of some catechins in urine and fecal samples but decreased the levels of their metabolites. These results suggest that antibiotics eliminated gut microbes and increased the bioavailabilities of these tea catechins. In a second study, mice were given different concentrations of green tea infusions as the drinking fluid. The plasma levels of EGCG and (-)-epicatechin-3-gallate (ECG) at day 112 were significantly lower than those at day 5. The urine levels of EGCG and ECG increased in the first 4 or 5 days, and then decreased to much lower levels at day 23 and beyond. In contrast, the levels of (-)-epigallocatechin and (-)-epicatechin showed a trend of increase during the 112-day experiment, likely owing to microbial hydrolysis of EGCG and ECG. Both sets of experiments support the idea that the degradation of EGCG and ECG by gut microbiota decreases their bioavailabilities. © 2018 BioFactors, 2018.

Intake of stigmasterol and ß-sitosterol alters lipid metabolism and alleviates NAFLD in mice fed a high-fat western-style diet.

OBJECTIVE: To investigate and compare the effects of two common dietary phytosterols, stigmasterol and ß-sitosterol, in altering lipid metabolism and attenuating nonalcoholic fatty liver disease (NAFLD). METHODS: Stigmasterol and ß-sitosterol were administered to mice at 0.4% in a high-fat western-style diet (HFWD) for 17 weeks. RESULTS: Stigmasterol and ß-sitosterol significantly ameliorated HFWD-induced fatty liver and metabolic abnormalities, including elevated levels of hepatic total lipids, triacylglycerols, cholesterol and liver histopathology. Both phytosterols decreased the levels of intestinal bile acids, accompanied by markedly increased fecal lipid levels. In addition, they altered the expression of genes involved in lipid metabolism. ß-Sitosterol was less effective in affecting most of these parameters. Lipidomic analysis of liver and serum samples showed that stigmasterol prevented the HFWD-induced elevation of some di- and triacylglycerol species and lowering of some phospholipid species. Stigmasterol also decreased serum levels of ceramides. CONCLUSION: Stigmasterol and ß-sitosterol, at a dose corresponding to that suggested for humans by the FDA for lowering cholesterol levels, are shown to alleviate HFWD-induced NAFLD. Stigmasterol was more effective than ß-sitosterol, possibly because of its suppression of hepatic lipogenic gene expression and modulation of circulating ceramide levels.

Effects of Stigmasterol and ß-Sitosterol on Nonalcoholic Fatty Liver Disease in a Mouse Model: A Lipidomic Analysis.

To study the effects of stigmasterol and ß-sitosterol on high-fat Western diet (HFWD)-induced nonalcoholic fatty liver disease (NAFLD), lipidomic analyses were conducted in liver samples collected after 33 weeks of the treatment. Principal component analysis showed these phytosterols were effective in protecting against HFWD-induced NAFLD. Orthogonal projections to latent structures-discriminate analysis (OPLS-DA) and S-plots showed that triacylglycerols (TGs), phosphatidylcholines, cholesteryl esters, diacylglycerols, and free fatty acids (FFAs) were the major lipid species contributing to these discriminations. The alleviation of NAFLD is mainly associated with decreases in hepatic cholesterol, TGs with polyunsaturated fatty acids, and alterations of free hepatic FFA. In conclusion, phytosterols, at a dose comparable to that suggested for humans by the FDA for the reduction of plasma cholesterol levels, are shown to protect against NAFLD in this long-term (33-week) study.

Crosstalk between bone marrow-derived myofibroblasts and gastric cancer cells regulates cancer stemness and promotes tumorigenesis.

Bone marrow-derived cells have important roles in cancer development and progression. Our previous studies demonstrated that murine bone marrow-derived myofibroblasts (BMFs) enhanced tumor growth. In this study, we investigated the mechanisms of BMF actions. We found that co-injection of BMFs with gastric cancer cells markedly promoted tumorigenesis. Co-cultured BMFs or BMF-conditioned medium (BMF-CM) induced the formation of spheres, which expressed stem cell signatures and exhibited features of self-renewal, epithelial-to-mesenchymal transition and tumor initiation. Furthermore, CD44+ fractions in spheres were able to initiate tumorigenesis and re-establish tumors in serially passaged xenografts. In co-culture systems, BMFs secreted high levels of murine interleukin-6 (IL-6) and hepatocyte growth factor (HGF), whereas cancer cells produced high level of transformation growth factor-ß1 (TGF-ß1). BMF-CM and IL-6 activated BMFs to produce mHGF, which activated signal transducer and activator of transcription 3 (STAT3) and upregulated TGF-ß1 in human cancer cells. In return, cancer cell-CM stimulated BMFs to produce IL-6, which was inhibited by anti-TGF-ß1 neutralizing antibody. Blockade of HGF/Met, Janus kinase 2 (JAK2)/STAT3 and TGF-ß1 signaling by specific inhibitors inhibited BMF-induced sphere formation. STAT3 knockdown in cancer cells also inhibited BMF-induced sphere formation and tumorigenesis. Moreover, TGF-ß1 overexpression in cancer cells was co-related with IL-6 and HGF overexpression in stromal cells in human gastric cancer tissues. Our results show that BMF-derived IL-6/HGF and cancer cell-derived TGF-ß1 mediate the interactions between BMFs and gastric cancer cells, which regulate cancer stemness and promote tumorigenesis. Targeting inhibition of the interactions between BMFs and cancer cells may be a new strategy for cancer therapy.

δ- and γ-tocopherols, but not α-tocopherol, inhibit colon carcinogenesis in azoxymethane-treated F344 rats.

The cancer preventive activity of vitamin E has been extensively discussed, but the activities of specific forms of tocopherols have not received sufficient attention. Herein, we compared the activities of δ-tocopherol (δ-T), γ-T, and α-T in a colon carcinogenesis model. Male F344 rats, seven weeks old, were given two weekly subcutaneous injections of azoxymethane (AOM) each at a dose of 15 mg/kg body weight. Starting 1 week before the AOM injection, the animals were maintained on a modified AIN76A diet, or the same diet containing 0.2% of δ-T, γ-T, α-T, or a γ-T-rich mixture of tocopherols (γ-TmT), until the termination of the experiment at 8 weeks after the second AOM injection. δ-T treatment showed the strongest inhibitory effect, decreasing the numbers of aberrant crypt foci by 62%. γ-T and γ-TmT were also effective, but α-T was not. Immunohistochemical analysis showed that δ-T and γ-T treatments reduced the levels of 4-hydroxynonenal and nitrotyrosine and the expression of cyclin D1 in the colon, preserved the expression of PPAR-γ, and decreased the serum levels of prostaglandin E2 and 8-isoprostane. Supplementation with 0.2% δ-T, γ-T, or α-T increased the respective levels of tocopherols and their side-chain degradation metabolites in the serum and colon tissues. Rather high concentrations of δ-T and γ-T and their metabolites were found in colon tissues. Our study provides the first evidence for the much higher cancer preventive activity of δ-T and γ-T than α-T in a chemically induced colon carcinogenesis model. It further suggests that δ-T is more effective than γ-T.

Dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced prostate carcinogenesis in CYP1A-humanized mice.

To develop a relevant mouse model for prostate cancer prevention research, we administered a dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to CYP1A-humanized mice. In comparison with mouse Cyp1a2, human CYP1A2 preferentially activates PhIP to a proximate carcinogen. Following a single oral dose of PhIP (200 mg/kg body weight), we observed inflammation, atrophy of acini, low-grade prostatic intraepithelial neoplasia (PIN; after 20 weeks), and high-grade PIN (HgPIN; after 30 to 50 weeks) in dorsolateral, ventral, and coagulating anterior prostate glands of these mice. These lesions were androgen receptor positive and featured the loss of expression of the basal cell marker p63 and the tumor suppressor PTEN. Similar to human prostate carcinogenesis, glutathione S-transferase P1 (GSTP1) expression was lost or partially lost in HgPIN. E-Cadherin expression was also lost in HgPIN. The expression of DNA methyltransferase 1 was elevated, possibly to enhance promoter hypermethylation for the silencing of GSTP1 and E-cadherin. Prostate carcinogenesis was promoted by a high-fat stress diet, resulting in HgPIN that developed earlier and in advanced lesions displayed features consistent with carcinoma in situ. This dietary carcinogen-induced prostate cancer model, recapitulating important features of early human prostate carcinogenesis, constitutes a new experimental system for prostate cancer research.

δ-tocopherol is more active than α - or γ -tocopherol in inhibiting lung tumorigenesis in vivo.

In contrast to strong epidemiologic, preclinical, and secondary clinical evidence for vitamin E (tocopherols) in reducing cancer risk, large-scale clinical cancer-prevention trials of α-tocopherol have been negative. This vexing contrast helped spur substantial preclinical efforts to better understand and improve the antineoplastic activity of tocopherol through, for example, the study of different tocopherol forms. We previously showed that the γ-tocopherol-rich mixture (γ-TmT) effectively inhibited colon and lung carcinogenesis and the growth of transplanted lung-cancer cells in mice. We designed this study to determine the relative activities of different forms of tocopherol in a xenograft model, comparing the anticancer activities of δ-tocopherol with those of α- and γ-tocopherols. We subcutaneously injected human lung cancer H1299 cells into NCr nu/nu mice, which then received α-, γ-, or δ-tocopherol or γ-TmT in the diet (each at 0.17% and 0.3%) for 49 days. δ-Tocopherol inhibited tumor growth most strongly. γ-Tocopherol and γ-TmT (at 0.3%) also inhibited growth significantly, but α-tocopherol did not. δ-Tocopherol also effectively decreased oxidative DNA damage and nitrotyrosine formation and enhanced apoptosis in tumor cells; again, γ-tocopherol also was active in these regards but less so, and α-tocopherol was not. Each supplemented diet increased serum levels of its tocopherol - up to 45 µmol/L for α-tocopherol, 9.7 µmol/L for γ-tocopherol, and 1.2 µmol/L for δ-tocopherol; dietary γ- or δ-tocopherol, however, decreased serum α-tocopherol levels, and dietary α-tocopherol decreased serum levels of γ-tocopherol. Each dietary tocopherol also increased its corresponding side-chain-degradation metabolites, with concentrations of δ-tocopherol metabolites greater than γ-tocopherol and far greater than α-tocopherol metabolites in serum and tumors. This study is the first in vivo assessment of δ-tocopherol in tumorigenesis and shows that δ-tocopherol is more active than α- or γ-tocopherol in inhibiting tumor growth, possibly through trapping reactive oxygen and nitrogen species and inducing apoptosis; δ-tocopherol metabolites could contribute significantly to these results.