Anne Constable

Cafestol and kahweol, two coffee specific diterpenes with anticarcinogenic activity.

Epidemiological studies have found an inverse association between coffee consumption and the risk of certain types of cancers such as colorectal cancers. Animal data support such a chemopreventive effect of coffee. Substantial research has been devoted to the identification of coffee components that may be responsible for these beneficial effects. In animal models and cell culture systems, the coffee diterpenes cafestol and kahweol (C+K) were shown to produce a broad range of biochemical effects resulting in a reduction of the genotoxicity of several carcinogens including 7,12-dimethylbenz[a]anthracene (DMBA), aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Different mechanisms appear to be involved in these chemoprotective effects: an induction of conjugating enzymes (e.g. glutathione S-transferases, glucuronosyl S-transferases), an increased expression of proteins involved in cellular antioxidant defense (e.g. gamma-glutamyl cysteine synthetase and heme oxygenase-1) and an inhibition of the expression and/or activity of cytochromes P450 involved in carcinogen activation (e.g. CYP2C11, CYP3A2). In animal models, the C+K-mediated induction of conjugating and antioxidant enzymes has been observed in hepatic, intestinal and kidney tissues. In the small intestine, these inductions were shown to be mediated by Nrf2-dependent transcriptional activation. In vitro investigations obtained in cell cultures of human origin indicate that the effects and mechanisms observed in animal test systems with C+K are likely to be of relevance for humans. In human liver epithelial cell lines transfected to express AFB(1)-activating P450s, C+K treatment resulted in a reduction of AFB(1)-DNA binding. This protection was correlated with an induction of GST-mu, an enzyme known to be involved in AFB(1) detoxification. In addition, C+K was found to inhibit P450 2B6, one of the human enzymes responsible for AFB(1) activation. Altogether, the data on the biological effects of C+K provide a plausible hypothesis to explain some of the anticarcinogenic effects of coffee observed in human epidemiological studies and in animal experiments.

Cutaneous lycopene and β-carotene levels measured by resonance Raman spectroscopy: high reliability and sensitivity to oral lactolycopene deprivation and supplementation.

Carotenoids, naturally occurring lipophilic micronutrients, possess an antioxidant activity associated with protection from damage induced by free radicals. The present study investigated an innovative non-invasive method to measure cutaneous levels of lycopene and beta-carotene and to monitor the distribution of orally administered lactolycopene in human skin and plasma. A double-blind placebo-controlled randomized study was performed in 25 volunteers, who were under a lycopene-deprived diet (4 weeks prior to study until end of the study) and orally received either lactolycopene or placebo for 12 weeks. Skin and plasma levels of lycopene and beta-carotene were monitored monthly using Raman spectroscopy and HPLC, respectively. Cutaneous levels of lycopene and beta-carotene monitored by resonance Raman spectroscopy showed high reliability. Irrespective of the investigated area, cutaneous levels were sensitive to lycopene deprivation and to oral supplementation; the forehead showed the closest correlation to lycopene variation in plasma. Plasma and skin levels of lycopene were both sensitive to oral intake of lactolycopene and, interestingly, also skin levels of beta-carotene. Thus, oral supplementation with lycopene led to an enrichment of beta-carotene in human skin, possibly due to the fact that carotenoids act in the skin as protection chains, with a natural protection against free radicals.

Interspecies differences in metabolism of heterocyclic aromatic amines by rat and human P450 1A2

The catalytic efficiences of cytochrome P450 (P450)-mediated N-oxidation of the 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by recombinant human P450 1A2 were 10-19-fold higher than rat P450 1A2, while methoxyresorufin O-demethylation activity was comparable for both P450s. Similar findings were observed with rat and human liver microsomal samples. Interspecies differences in P450 enzyme expression and catalytic activities may be significant and must be considered when assessing human health risk.

Regulatory control of genetically modified (GM) foods: likely developments

The placing of genetically modified (GM) crops on the European market requires a regulatory approval supported by a thorough safety evaluation. This approach has been applied to all GM crops presently on the market. Despite this stringent process there has been an increasing public concern about the impact of GM foods on human health and the environment. In this context, regulatory control may develop in several directions. One response to the public concern is to strengthen the data requirements for the risk assessment process. Several avenues have been proposed. They include the application of technologies such as proteomics and metabolomics to assess unintended changes, and the development of predictive methods to evaluate allergenicity. Obligations for post-launch surveillance have appeared in regulations. Criteria are required to define when and why such approaches are necessary. Significant challenges including feasibility and validation of the methods, and safety relevance of the data generated will have to be addressed before any general application of these new approaches. Effective monitoring requires the ability to identify the presence of GM products and trace their origin. Traceability and labeling are therefore important developments in the GM food regulatory arena. Both require the development of reliable analytical detection tools.

The effects of coffee on enzymes involved in metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine in rats

The effects of coffee on the metabolism and genotoxicity of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were investigated. Coffee diminished the bacterial mutagenicity of PhIP in the Ames reversion assay through inhibition of cytochrome P450 1A2 (CYP1A2), a key enzyme involved in the metabolic activation of PhIP. When given as part of the diet (0, 1 or 5% w/w) to male Fischer-344 rats for 2 weeks, coffee affected the expression of hepatic enzymes involved in PhIP metabolism. Coffee increased the expression of CYP1A2 by 16-fold in the 5% coffee-treated group, and approximately half of this inductive effect was attributed to caffeine. Coffee also increased the expression of enzymes involved in the detoxication of PhIP. A 2-fold increase in expression of glutathione S-transferase alpha was observed, UDP-glucuronosyl transferase (UGTs) activities of p-nitrophenol increased 2-fold, while N(2)-and N3-glucuronidation of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP) increased by 1.3-fold in the 5% coffee-treated over the control group. The amount of PhIP (0.75 mg/kg, 24 h) eliminated in urine as the N(2)-and N3-glucuronide conjugates of HONH-PhIP increased by 1.8- and 2.5-fold, respectively, in the 5% coffee-treated group over control rats, suggesting either increased rates of N-oxidation of PhIP or N-glucuronidation of HONH-PhIP. Despite the strong induction of CYP1A2, there was no increase in PhIP-DNA adduct formation in colon and pancreas while liver adducts decreased by 50% over control animals. These data suggest that the effect of coffee on inhibition of PhIP N-oxidation and ensuing DNA damage is more important in vivo than its effect on induction of PhIP N-hydroxylation.

Dietary strategies for improving iron status: balancing safety and efficacy

In light of evidence that high-dose iron supplements lead to a range of adverse events in low-income settings, the safety and efficacy of lower doses of iron provided through biological or industrial fortification of foodstuffs is reviewed. First, strategies for point-of-manufacture chemical fortification are compared with biofortification achieved through plant breeding. Recent insights into the mechanisms of human iron absorption and regulation, the mechanisms by which iron can promote malaria and bacterial infections, and the role of iron in modifying the gut microbiota are summarized. There is strong evidence that supplemental iron given in nonphysiological amounts can increase the risk of bacterial and protozoal infections (especially malaria), but the use of lower quantities of iron provided within a food matrix, ie, fortified food, should be safer in most cases and represents a more logical strategy for a sustained reduction of the risk of deficiency by providing the best balance of risk and benefits. Further research into iron compounds that would minimize the availability of unabsorbed iron to the gut microbiota is warranted.

Considering new methodologies in strategies for safety assessment of foods and food ingredients

Toxicology and safety assessment are changing and require new strategies for evaluating risk that are less depending on apical toxicity endpoints in animal models and relying more on knowledge of the mechanism of toxicity. This manuscript describes a number of developments that could contribute to this change and implement this in a stepwise roadmap that can be applied for the evaluation of food and food ingredients. The roadmap was evaluated in four case studies by using literature and existing data. This preliminary evaluation was shown to be useful. However, this experience should be extended by including examples where experimental work needs to be included. To further implement these new insights in toxicology and safety assessment for the area of food and food ingredients, the recommendation is that stakeholders take action in addressing gaps in our knowledge, e.g. with regard to the applicability of the roadmap for mixtures and food matrices. Further development of the threshold of toxicological concern is needed, as well as cooperation with other sectors where similar schemes are under development. Moreover, a more comprehensive evaluation of the roadmap, also including the identification of the need for in vitro experimental work is recommended.

Pre-clinical safety assessment of the synthetic human milk, nature-identical, oligosaccharide Lacto-N-neotetraose (LNnT).

Lacto-N-neotetraose (LNnT) is a tetrasaccharide naturally occurring in human breast milk, but not in cows milk. The safety data generated on a potential new LNnT ingredient produced by chemical synthesis is presented. Standard in vitro genotoxicity tests were performed. LNnT was also administered via gavage in 14-, 28- and 90-day studies at levels corresponding to 0 (control), 1000, 2500 and 5000 mg/kg bw/day in juvenile rats. Fructooligosaccharide (FOS) currently approved for use in infant formulae was used as a reference control at one dose level of 5000 mg/kg bw/day. LNnT was non-mutagenic in in vitro assays. Oral administration up to 5000 mg/kg bw/day to rats over 90 days was not associated with any adverse effects, based on clinical observations, body weight gain, feed consumption, clinical pathology, organ weights and histopathology findings. Regarding gastrointestinal effects, LNnT was better tolerated than FOS during the first 2 weeks of treatment. A No Observed Adverse Effect Level (NOAEL) of 5000 mg/kg bw/day for both male and female rats was identified for LNnT when administered by gavage for 90 days. These findings in the juvenile rat support the safety of LNnT for possible use in infant foods and allow further investigation in clinical studies.

N,N-dimethylpiperidinium (mepiquat) Part 2. Formation in roasted coffee and barley during thermal processing

Previous work in model systems has demonstrated that mepiquat can be formed under typical roasting conditions from the amino acid lysine via the Maillard reaction and trigonelline, the latter alkaloid serving as a methyl donor. This study shows for the first time that mepiquat is formed in low mg kg–1 amounts during the coffee roasting process and consequently can be detected in roast and ground as well as soluble coffee up to levels of 1.4 mg kg–1. Darker roast coffees contain relatively higher amounts of mepiquat versus light roasted beans, with an excellent correlation of mepiquat formation to roast colour (r2 = 0.99) in robusta beans. A survey of 20 of the major green coffee origins (robusta and arabica coffees) showed the absence of mepiquat (<0.005 mg kg–1). Preliminary studies indicate that mepiquat is not formed during processing (thermal treatment) in most of the cereal-based foods such as pizza and ready-to-eat cereals, but was detected in barley after roasting (0.64 mg kg–1). Mepiquat can therefore be considered a process-induced compound formed from natural constituents during the roasting process. Even considering a high intake of seven cups per day of soluble coffee containing 1.4 mg kg–1 mepiquat in the coffee powder (the highest amount measured in this study), the resulting intake would exhaust less than 0.2% of the ADI of mepiquat.

Using urinary solubility data to estimate the level of safety concern of low levels of melamine (MEL) and cyanuric acid (CYA) present simultaneously in infant formulas

Melamine (MEL) and cyanuric acid (CYA) may occur simultaneously in milk products. There is no health based guidance value for the mixture of MEL+CYA. Limited toxicological data indicate that MEL+CYA toxicity occurs at levels lower than the toxic doses of the single compounds. The key adverse effect of MEL+CYA is the formation of crystals in the urinary tract, which is dependent on the solubility of the MEL+CYA complex. Urinary concentrations resulting from oral doses of MEL+CYA and MEL alone have been calculated from published data from animal studies. A human exposure scenario assuming consumption of infant formula contaminated at a level of 1 ppm of MEL and CYA each (2 ppm of MEL+CYA) was also analyzed. Margins of more than two orders or magnitude were observed between estimated urine concentrations known to be without detectable effects in rats and calculated human urine concentrations. Because the hazard is related to the physico-chemical characteristics of the mixture, there would be a negligible concern associated with crystal formation if the urinary concentration of the complex is within the solubility range. The solubility of MEL+CYA was higher in urine than in water. A strong pH-dependency was observed with the lowest solubility found at pH 5-5.5. The calculated human urinary concentration was about 30 times less than the solubility limit for MEL+CYA in adult human urine. Altogether, these data provide preliminary evidence suggesting that the presence of 1 ppm of MEL and CYA each in infant formula is unlikely to be of significant health concern.