Ole Vang

What Is New for an Old Molecule? Systematic Review and Recommendations on the Use of Resveratrol

Background Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark. Methodology Literature search in databases as PubMed and ISI Web of Science in combination with manual search was used to answer the following five questions: 1Can resveratrol be recommended in the prevention or treatment of human diseases?; 2Are there observed “side effects” caused by the intake of resveratrol in humans?; 3What is the relevant dose of resveratrol?; 4What valid data are available regarding an effect in various species of experimental animals?; 5Which relevant (overall) mechanisms of action of resveratrol have been documented? Conclusions/Significance The overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.

Challenges of Translating Basic Research Into Therapeutics: Resveratrol as an Example

Basic science literature abounds with molecules that promise to ameliorate almost any disease, from curing cancer to slowing the aging process itself. However, most of these compounds will never even be evaluated in humans, let alone proven effective. Here, we use resveratrol as an example to highlight the enormous difficulties in understanding pharmacokinetics, determining side effects, and, ultimately, establishing mechanisms of action for a natural compound. Despite extensive interest and effort, and continuing promising results from basic science groups, very little is known even today about the effects of resveratrol in humans. Part of the problem is the unattractiveness of natural compounds to large, well-funded companies that could run clinical trials because developing their own molecules affords much greater protection for their intellectual property. In fact, selling unpatentable material motivates smaller nutraceutical companies to complicate the scientific problem even more--each creates its own proprietary blend, making it extremely difficult to compare their data with those of other companies, or of academic labs using pure compounds. But even beyond these problems lies a deeper one; resveratrol, and almost every natural compound, is likely to have many clinically relevant targets with different dose-response profiles, tissue distributions, and modifiers. Tackling this type of problem efficiently, and even beginning to address the spectrum of other molecules with claimed benefits, is likely to require the development of new paradigms and approaches. Examples include better molecular modeling to predict interactions, large-scale screens for toxic or other common effects, affinity-based methods to identify drug-interacting proteins, and better synthesis of existing data, including legislation to promote the release of trial results, and tracking of voluntary supplement usage. The evidence for benefits of resveratrol in humans remains too sparse to be conclusive; yet, the limited data that are available, combined with a growing list of animal studies, provide a strong justification for further study.

Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon

Ingestion of broccoli or other cruciferous vegetables inhibits the induction of cancer by chemicals and modifies some cytochrome P-450 enzyme activities. The effect of dietary broccoli on the levels of P450IA and IIB mRNA and proteins in rat liver and colon has been studied. Rats were fed a ten percent broccoli diet for 7 days. The expression of the cytochrome P-450 forms was altered to a different extent in the liver and colon. The level of total P450IA mRNA in the liver was increased by the broccoli together with the P450IA1 and IA2 proteins. Colonic P450IA1 mRNA and protein were induced by the broccoli diet, whereas only P450IA2 protein and not mRNA was detectable in colon, but the protein level was unaffected by the broccoli diet. Liver P450IIB and IIE1 proteins were increased by the broccoli diet, whereas the level of P450IIB mRNAs was not affected. In contrast, the P450IIB mRNA levels were reduced but the protein levels were increased in colon and we suggest that a feedback mechanism caused the decrease of the P450IIB mRNAs levels. Because the ratio between activation and deactivation may be an important risk determinant, we conclude that the protective effect of the broccoli diet on chemically induced tumors in rodents may be caused by the broccoli-induced changes in P450IA and IIB associated enzyme activities.

Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

Abstract.  Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL‐60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration‐dependent manner. The IC50 value was 15 µm in both HL‐60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 µm for HL‐60 cells and 60 µm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL‐60 treated and HepG2‐treated cells. Apoptosis was also stimulated by resveratrol in a concentration‐dependent manner in HL‐60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration‐ and time‐dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

Modulation of cytochrome P-450 and glutathione S-transferase isoform expression in vivo by intact and degraded indolyl glucosinolates.

Various dietary substances modulate the xenobiotic metabolism and may thereby protect against toxicity and carcinogenicity of food toxins. The effects of pure indolyl glucosinolates, which are present in cruciferous vegetables, on induction of specific cytochrome P-450 (CYP) and glutathione S-transferase (GST) isoforms have not been studied previously. In the present study, glucobrassicin (GB) and neoglucobrassicin (NeoGB) were purified from broccoli by use of a single-column method. Furthermore, a mixture containing 48% GB, 36% NeoGB, and 16% 4-methoxyglucobrassicin was obtained. The modulatory effects of the pure GB, NeoGB, and the mixture on activities and levels of hepatic CYP 1A, 2B1/2, and 2E1 and alpha- and mu-GST isoforms were investigated in male Wistar rats. The indolyl mixture was the most powerful and NeoGB the weakest inducer of microsomal hepatic CYP 1A1 protein and 7-ethoxyresorufin O-deethylase activity. Furthermore, intact indolyl glucosinolates were more powerful inducers than the in vitro myrosinase-degraded indolyl glucosinolates. The hepatic 7-methoxyresorufin O-deethylase activities, but not CYP 1A2 protein, were induced by pure GB, whereas the mixture and NeoGB showed only minor effects. Neither CYP 2B1/2 nor 2E1 was induced by the indolyl glucosinolates. None of the hepatic GST subunits analyzed, rGST A1/2, A3, or M3, was induced significantly by the purified indolyl glucosinolates.

N-Methoxyindole-3-Carbinol Is a More Efficient Inducer of Cytochrome P-450 1A1 in Cultured Cells Than Indol-3-Carbinol

The well-documented reduction of cancer risk by high dietary cruciferous vegetable intake may in part be caused by modulation of cytochrome P-450 (CYP) expression and activity by indoles. The purpose of the present experiments was to study the mechanism of CYP 1A1 induction by N-methoxyindole-3-carbinol (NI3C) in cultured cells and to compare the CYP-inducing potential of NI3C and indole- 3-carbinol (I3C) administered to rats. NI3C induced 7-ethoxyresorufin-O-deethylase (EROD) activity in Hepa-1c1c7 cells in a concentration-dependent manner with 10-fold higher efficiency than I3C. Inasmuch as NI3C induced binding of the aryl hydrocarbon receptor (AhR) to the dioxin-responsive element and induced expression of endogenous CYP 1A1 mRNA and an AhR-responsive chloramphenicol acetyl transferase construct, we conclude that NI3C can activate the AhR. Besides the induction of CYP 1A1, we observed an inhibition of EROD activity, with a concentration causing 50% inhibition of 6 μM. Oral administration of NI3C at 570 μmol/kg body wt to male Wistar rats increased the hepatic CYP 1A1 and 1A2 protein levels, as well as the EROD and 7-methoxyresorufin O-demethylase activities at 8 and 24 hours after administration, but the responses were less pronounced than after administration of I3C at 570 μmol/kg body wt. Furthermore, NI3C did not induce hepatic 7-pentoxyresorufin O-depentylase activity, as I3C did. Ascorbigen, another indolylic compound formed during degradation of glucobrassicin in the presence of ascorbic acid, was tested in the same animal model, and ascorbigen only weakly induced hepatic CYP 1A1 and 1A2, but not CYP 2B1/2. In conclusion, NI3C is a more efficient inducer of CYP 1A1 in cultured cells than I3C but is less active when administered to rodents.

What is new for resveratrol? Is a new set of recommendations necessary?

Numerous scientific papers have suggested health‐promoting effects of resveratrol, including claims in the prevention of diseases such as coronary heart disease, diabetes, and cancer. Therefore, it was proposed that the scientific community needed to express recommendations on the human use of resveratrol. Such recommendations were formulated after the first international resveratrol conference in Denmark, Resveratrol2010. The working group stated that the evidence was “not sufficiently strong to justify recommendation for the chronic administration of resveratrol to human beings, beyond the dose which can be obtained from dietary sources.” It was a disappointing conclusion relative to the positive claims about the therapeutic potential of resveratrol made by the media. However, since 2010, results from the first clinical trials on resveratrol have been made available. Because of these emerging results, it is necessary to formulate updated versions of the recommendations.

Challenges in Analyzing the Biological Effects of Resveratrol

The suggested health effects (e.g., disease prevention) of dietary bioactive compounds such as resveratrol are challenging to prove in comparison to man-made drugs developed for therapeutic purposes. Dietary bioactive compounds have multiple cellular targets and therefore have a variety of biological effects. Extrapolating the biological effects of dietary compounds from in vitro and in vivo animal experiments to humans may lead to over- or under-estimation of the effect and role of these compounds. The present paper will discuss a few of these challenges and suggest directions for future research. Questions we address include: (1) Is the combinatorial effect of resveratrol and other compounds real? (2) What are the real and relevant doses of resveratrol after administration? and (3) Is it possible to estimate the preventive effect of resveratrol by clinical trials using standard experimental designs? The examples concerning resveratrol taken from the scientific literature are mainly from 2010 and later. The challenges pointed out in this review are similar to most naturally occurring bioactive compounds.

Combined effects of complex mixtures of potentially anti-carcinogenic compounds on antioxidant enzymes and carcinogen metabolizing enzymes in the rat

The anti-carcinogenic activity of dietary fruit and vegetables observed in several epidemiological and experimental animal studies is likely to be an effect of the combined exposure to a large number of substances acting together. This is plausible, as these compounds are present simultaneously in a diet containing vegetables and fruit. Further, some compounds have been experimentally demonstrated to modify several mechanisms involved in carcinogenesis. The effect of combined exposure is demonstrated in the present article, summarizing the effects of a complex mixture of anti-carcinogenic substances (from broccoli) on different antioxidative defense enzymes and on cytochrome P-450 activities in rat liver, kidney and colon. The responses were related to the levels of different specific glucosinolates.