Toxicologic evaluation of a novel, highly soluble biotin salt, magnesium biotinate.

A novel, highly soluble biotin salt, magnesium biotinate (MgB), was assessed for general and genetic toxicity using several toxicologic tests. This battery of tests included in vitro bacterial reverse mutation test, in vitro mammalian micronucleus assay, and oral acute, 14-day, and 90-day repeat-dose toxicity in Sprague-Dawley (SD) rats. The results of the in vitro studies indicate that MgB is not mutagenic, clastogenic, or aneugenic. The acute oral toxicity study established an LD50 ≥ 5000 mg MgB/kg. In the 14-day oral toxicity study, doses of MgB up to 2500 mg MgB/kg/day produced no clinical signs or mortality. In the 90-day oral toxicity study, administration of 600 mg MgB/kg/day resulted in no clinical signs and was determined to be the no-observed-adverse-effect-level (NOAEL), which equates to 39 g biotin/day for a 70 kg human. Since MgB is composed of 93% biotin, the 600 mg NOAEL equates to approximately 1.3 million times the current recommended daily allowance of 30 µg biotin/day and 3900 times supplement levels of 10 mg biotin/day. Based on the toxicologic profile and lack of findings in various in vitro and in vivo studies, MgB may be considered safe for long-term human use.

δ- and γ-tocopherols inhibit phIP/DSS-induced colon carcinogenesis by protection against early cellular and DNA damages.

Tocopherols, the major forms of vitamin E, are a family of fat-soluble compounds that exist in alpha (α-T), beta (ß-T), gamma (γ-T), and delta (δ-T) variants. A cancer preventive effect of vitamin E is suggested by epidemiological studies. However, past animal studies and human intervention trials with α-T, the most active vitamin E form, have yielded disappointing results. A possible explanation is that the cancer preventive activity of α-T is weak compared to other tocopherol forms. In the present study, we investigated the effects of δ-T, γ-T, and α-T (0.2% in diet) in a novel colon cancer model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by dextran sodium sulfate (DSS)-induced colitis in CYP1A-humanized (hCYP1A) mice. PhIP/DSS treatments induced multiple polypoid tumors, mainly tubular adenocarcinomas, in the middle to distal colon of the hCYP1A mice after 10 wk. Dietary supplementation with δ-T and γ-T significantly reduced colon tumor formation and suppressed markers of oxidative and nitrosative stress (i.e., 8-oxo-dG and nitrotyrosine) as well as pro-inflammatory mediators (i.e., NF-κB p65 and p-STAT3) in tumors and adjacent tissues. By administering δ-T at different time periods, we obtained results suggesting that the inhibitory effect of δ-T against colon carcinogenesis is mainly due to protection against early cellular and DNA damages caused by PhIP. α-T was found to be ineffective in inhibiting colon tumors and less effective in attenuating the molecular changes. Altogether, we demonstrated strong cancer preventive effects of δ-T and γ-T in a physiologically relevant model of human colon cancer. © 2016 Wiley Periodicals, Inc.

From the Cover: PhIP/DSS-Induced Colon Carcinogenesis in CYP1A-Humanized Mice and the Possible Role of Lgr5+ Stem Cells.

In the past decades, experimental rodent models developed to study the pathogenesis of human colorectal cancer (CRC) generally employed synthetic chemical carcinogens or genetic manipulation. Our lab, in order to establish a more physiologically relevant CRC model, recently developed a colon carcinogenesis model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and promoted by dextran sodium sulfate (DSS)-induced colitis in the cytochrome P450 1A-humanized (hCYP1A) mice. The resulting colon tumors shared many histologic and molecular features of human colon cancer. In this study, we characterized the early stages of PhIP/DSS-induced colon carcinogenesis. We found that PhIP/DSS treatments caused rapid destruction of the colon mucosa with severe inflammation, followed by the presence of reactive changes and low-grade dysplastic lesions, and then manifestation of high-grade dysplastic lesions and finally adenocarcinomas. Molecular analysis of the early time-points (ie, days 1, 3, 7, 11, 14, and 21 after DSS exposure) indicates Ctnnb1/ß-catenin mutations and ß-catenin nuclear accumulation in the high-grade dysplastic lesions, but not low-grade dysplastic lesions or adjacent normal tissues. In addition, we investigated the role of Lgr5+ colon stem cells in the PhIP/DSS-induced colon carcinogenesis and found the presence of Lgr5-enhance green fluorescent protein-expressing cells amidst some ulcerated mucosa, high-grade dysplastic lesions and adenocarcinomas, suggesting a possible role of Lgr5+ stem cells in this dietary carcinogen-induced, inflammation-promoted colon carcinogenesis model. Overall, the findings suggest that PhIP/DSS-induced colon carcinogenesis is likely initiated by dominant active Ctnnb1/ß-catenin mutation in residual epithelial cells, which when promoted by colitis, developed into high-grade dysplasia and adenocarcinoma.

Lessons learned from cancer prevention studies with nutrients and non-nutritive dietary constituents.

Epidemiological studies have observed the association between dietary patterns and the risk of certain types of cancer. Extensive studies have been conducted on the cancer preventive activities of constituents from food and beverages. While laboratory research has shown impressive and promising results, such promising cancer preventive activities have not been demonstrated in many human intervention trials. This article analyzes the major differences between these different types of studies and the limitations of these studies. Animal and cell line studies usually use optimal conditions in order to demonstrate the hypothesized effects, sometimes without considering the human relevance. On the other hand, some clinical trials were designed without a good understanding of the biochemical and pharmacological properties of the agents used. Lessons learned from these studies will be illustrated using vitamin E, ß-carotene and selenium as examples for nutrients, and green tea polyphenols as an example for non-nutritive dietary constituents. From the lessons learned, we believe that more interdisciplinary collaboration and integration of laboratory and human studies would effectively advance the field of cancer prevention.

Dietary tocopherols inhibit PhIP-induced prostate carcinogenesis in CYP1A-humanized mice.

Tocopherols, the major forms of vitamin E, exist as alpha-tocopherol (α-T), ß-T, γ-T and δ-T. The cancer preventive activity of vitamin E is suggested by epidemiological studies, but recent large-scale cancer prevention trials with high dose of α-T yielded disappointing results. Our hypothesis that other forms of tocopherols have higher cancer preventive activities than α-T was tested, herein, in a novel prostate carcinogenesis model induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a dietary carcinogen, in the CYP1A-humanized (hCYP1A) mice. Treatment of hCYP1A mice with PhIP (200 mg/kg b.w., i.g.) induced high percentages of mouse prostatic intraepithelial neoplasia (mPIN), mainly in the dorsolateral glands. Supplementation with a γ-T-rich mixture of tocopherols (γ-TmT, 0.3% in diet) significantly inhibited the development of mPIN lesions and reduced PhIP-induced elevation of 8-oxo-deoxyguanosine, COX-2, nitrotyrosine, Ki-67 and p-AKT, and the loss of PTEN and Nrf2. Further studies with purified δ-T, γ-T or α-T (0.2% in diet) showed that δ-T was more effective than γ-T or α-T in preventing mPIN formations and p-AKT elevation. These results indicate that γ-TmT and δ-T could be effective preventive agents of prostate cancer.

NNK-induced DNA methyltransferase 1 in lung tumorigenesis in A/J mice and inhibitory effects of (-)-epigallocatechin-3-gallate.

DNA methyltransferase 1 (DNMT1), a key enzyme mediating DNA methylation, is known to be elevated in various cancers, including the mouse lung tumors induced by the tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). However, it is not known whether DNMT1 expression is induced right after NNK treatment and how DNMT1 expression varies throughout lung tumorigenesis. In the present study, we found that administration of NNK to A/J mice caused elevation of DNMT1 in bronchial epithelial cells at Days 1, 3, and 14 after NNK treatment. DNMT1 elevation at Day 1 was accompanied by an increase in phospho-histone H2AX (γ-H2AX) and phospho-AKT (p-AKT). At Weeks 5 to 20, NNK-induced DNMT1 in lung tissues was in lower levels than the early stages, but was highly elevated in lung tumors at Week 20. In addition, the early induction of p-AKT and γ-H2AX as well as cleaved caspase-3 in NNK-treated lung tissues was not detected at Weeks 5 to 20 but was elevated in lung tumors. In concordance with DNMT1 elevation, promoter hypermethylation of tumor suppressor genes Cdh13, Prdm2, and Runx3 was observed in lung tissues at Day 3 and in lung tumors. Treatment by EGCG attenuated DNMT1, p-AKT, and γ-H2AX inductions at Days 1 and 3 and inhibited lung tumorigenesis.

Effects of Tea Catechins on Cancer Signaling Pathways.

The inhibitory activities of tea catechins against carcinogenesis and cancer cell growth have been demonstrated in a large number of laboratory studies. Many mechanisms for modulating cancer signaling and metabolic pathways have been proposed based on numerous studies in cell lines with (-)-epigallocatechin-3-gallate, the most abundant and active tea catechin. Nevertheless, the molecular basis for the proposed mechanisms and whether these mechanisms indeed contribute to the anticancer activities in vivo are not clearly known. This chapter reviews the basic redox properties of tea catechins, their binding to key enzymes and signal transduction proteins, and other mechanisms that lead to suppression of cell proliferation, increased apoptosis, and inhibition of angiogenesis. More weight is put on studies in vivo over experiments in vitro. It also discusses key issues involved in extrapolating results from cell line studies to mechanistic insights in vivo.

Gene regulation mediated by microRNAs in response to green tea polyphenol EGCG in mouse lung cancer.

BACKGROUND: Epigallocatechin-3-gallate (EGCG) has been demonstrated to inhibit cancer in experimental studies through its antioxidant activity and modulations on cellular functions by binding specific proteins. We demonstrated previously that EGCG upregulates the expression of microRNA (i.e. miR-210) by binding HIF-1α, resulting in reduced cell proliferation and anchorage-independent growth. However, the binding affinities of EGCG to HIF-1α and many other targets are higher than the EGCG plasma peak level in experimental animals administered with high dose of EGCG, raising a concern whether the microRNA regulation by HIF-1α is involved in the anti-cancer activity of EGCG in vivo. RESULTS: We employed functional genomic approaches to elucidate the role of microRNA in the EGCG inhibition of tobacco carcinogen-induced lung tumors in A/J mice. By analysing the microRNA profiles, we found modest changes in the expression levels of 21 microRNAs. By correlating these 21 microRNAs with the mRNA expression profiles using the computation methods, we identified 26 potential targeted genes of the 21 microRNAs. Further exploration using pathway analysis revealed that the most impacted pathways of EGCG treatment are the regulatory networks associated to AKT, NF-κB, MAP kinases, and cell cycle, and the identified miRNA targets are involved in the networks of AKT, MAP kinases and cell cycle regulation CONCLUSIONS: These results demonstrate that the miRNA-mediated regulation is actively involved in the major aspects of the anti-cancer activity of EGCG in vivo.