Pelle Thonning Olesen

Acrylamide exposure and incidence of breast cancer among postmenopausal women in the Danish Diet, Cancer and Health Study.

Acrylamide, a probable human carcinogen, is formed in several foods during high‐temperature processing. So far, epidemiological studies have not shown any association between human cancer risk and dietary exposure to acrylamide. The purpose of this study was to conduct a nested case control study within a prospective cohort study on the association between breast cancer and exposure to acrylamide using biomarkers. N‐terminal hemoglobin adduct levels of acrylamide and its genotoxic metabolite, glycidamide in red blood cells were analyzed (by LC/MS/MS) as biomarkers of exposure on 374 breast cancer cases and 374 controls from a cohort of postmenopausal women. The adduct levels of acrylamide and glycidamide were similar in cases and controls, with smokers having much higher levels (∼3 times) than nonsmokers. No association was seen between acrylamide‐hemoglobin levels and breast cancer risk neither unadjusted nor adjusted for the potential confounders HRT duration, parity, BMI, alcohol intake and education. After adjustment for smoking behavior, however, a positive association was seen between acrylamide‐hemoglobin levels and estrogen receptor positive breast cancer with an estimated incidence rate ratio (95% CI) of 2.7 (1.1–6.6) per 10‐fold increase in acrylamide‐hemoglobin level. A weak association between glycidamide hemoglobin levels and incidence of estrogen receptor positive breast cancer was also found, this association, however, entirely disappeared when acrylamide and glycidamide hemoglobin levels were mutually adjusted.

Generation of flavour compounds in fermented sausages-the influence of curing ingredients, Staphylococcus starter culture and ripening time.

The volatile profiles of fermented sausages made with either Staphylococcus xylosus or Staphylococcus carnosus starter cultures were studied with regard to the influence of salt concentration, ripening time and three different combinations of curing ingredients-nitrate, nitrite or nitrite/ascorbate. Emphasis was laid on volatile compounds originating from degradation of branched-chain amino acids. Volatile compounds were collected using dynamic headspace sampling and were identified by gas chromatography/mass spectrometry (GC/MS). Development in water activity, water loss and pH was monitored throughout maturation. Curing salts had a pronounced effect on the level of volatile compounds. In particular, curing with nitrate instead of nitrite resulted in a striking difference. Generally, nitrate increased the level of volatile compounds compared to nitrite, whereas ascorbate had only a small influence. The concentration level of NaCl had a considerable effect on the amount of volatile compounds but the effect was highly related to the ripening stage. Most compounds, but not all, increased in concentration as ripening proceeded. Major differences in the development of volatile compounds were observed depending on whether S. xylosus or S. carnosus were used as starter culture. In particular the effects of nitrate was much more predominant in the sausages made with S. carnosus than S.xylosus.

The influence of Debaryomyces hansenii and Candida utilis on the aroma formation in garlic spiced fermented sausages and model minces

The influence of the yeast starter cultures Debaryomyces hansenii and Candida utilis on fermented meat aroma was studied in model minces and in commercial-type fermented sausages. Volatile compounds from model minces and sausages were collected using diffusive and dynamic headspace sampling respectively and were identified by gas chromatography/mass spectrometry (GC/MS). A triangle test was carried out on the sausages to detect whether the yeast influenced the sausage odour. C. utilis demonstrated high metabolic activity in the model minces, producing several volatile compounds, in particularly esters. C. utilis also seemed to ferment the amino acids valine, isoleucine and leucine into compounds important for the aroma of sausages. D. hansenii on the contrary, had very little effect on the production of volatile compounds in the model minces. In the sausage experiment both yeast cultures died out before the ripening process ended and the sensory analysis showed only a slight difference between the sausages. A fungistatic test of the garlic powder added to the sausages indicated that garlic inhibits the growth of the yeast starter cultures.

Biomarkers of Human Exposure to Acrylamide and Relation to Polymorphisms in Metabolizing Genes

Acrylamide (AA) is formed in heat treated carbohydrate rich foods in the so-called Maillard reaction. AA is readily absorbed in the body and converted to glycidamide (GA) by epoxidation by the CYP2E1 (cytochrome P450 2E) enzyme. Both AA and GA may be detoxified through direct conjunction to glutathione by glutathione-S-transferases and GA by hydrolysis to glyceramide. Recently, we reported that biomarkers of AA exposure reflect intake of major food sources of AA; there were large interindividual variations in the blood ratio of GA-Hb/AA-Hb (GA- and AA-hemoglobin adducts). In this study we investigated whether the ratio of GA-Hb/AA-Hb in subjects could be related to polymorphic differences in genes coding for metabolizing enzymes CYP2E1, EPHX1 (microsomal epoxide hydrolase), GSTM1, GSTT1, and GSTP1, all being expected to be involved in the activation and detoxification of AA-associated adducts. We found significant associations between GSTM1 and GSTT1 genotypes and the ratio of GA-Hb/AA-Hb (p = 0.039 and p = 0.006, respectively). The ratio of GA-Hb/AA-Hb in individuals with the combined GSTM1- and GSTT1-null variants was significantly (p = 0.029) higher than those with the wild-type genotypes. Although the number of subjects was small, there were also significant associations with other combinations; CYP2E1 (Val179Val) plus GSTM1-null (p = 0.022); CYP2E1 (Val/Val), GSTM1-null plus GSTT1-null (p = 0.047); and CYP2E1 (Val/Val), GSTT1 null, EPHX1 (Tyr113Tyr) plus EPHX1 (His139Arg) (p = 0.018). Individuals with these combined genotypes had significantly higher blood ratio of GA-Hb/AA-Hb than other combinations. The observed associations correspond with what would be expected from the relative roles of these enzymes in activation and detoxification of AA, except for individuals with the EPHX1 (His139Arg) variant. The internal dose of genotoxic metabolite and also the concentration of AA in blood seem to be affected by these polymorphic genes. The genotypes and their combination may constitute useful biomarkers for the assessment of individual susceptibility to AA intake, and could add to the precision of epidemiological studies of dietary cancer.

Effect of ascorbate, nitrate and nitrite on the amount of flavour compounds produced from leucine by Staphylococcus xylosus and Staphylococcus carnosus

Resting cells of Staphylococcus xylosus and S. carnosus were incubated with ascorbate, nitrate and nitrite in defined reaction medium and their degradation of (3)H-labelled leucine into methyl-branched catabolites were studied using HPLC/radiometric detection. The experiments were carried out with and without addition of α-ketoglutarate. The main catabolic product of leucine degradation was 3-methylbutanoic acid but also small amounts of α-hydroxy isocaproic acid were produced. Nitrite addition lowered the concentration of 3-methylbutanoic acid for both Staphylococcus species and this effect was strongly amplified by ascorbate for S. xylosus but not for S. carnosus. For both species ascorbate alone had little if any effect. Also nitrate lowered the concentration of 3-methylbutanoic acid for S. xylosus. The concentration of α-hydroxy isocaproic acid was, however, increased by addition of nitrite and nitrate for S. xylosus. Addition of α-ketoglutarate generally increased the concentration of 3-methylbutanoic acid for both S. xylosus and S. carnosus.

Dietary exposure to volatile and non-volatile N-nitrosamines from processed meat products in Denmark.

Recent epidemiological studies show a positive association between cancer incidence and high intake of processed meat. N-nitrosamines (NAs) in these products have been suggested as one potential causative factor. Most volatile NAs (VNAs) are classified as probable human carcinogens, whereas the carcinogenicity for the majority of the non-volatile NA (NVNA) remains to be elucidated. Danish adults (15-75 years) and children (4-6 years) consume 20 g and 16 g of processed meat per day (95th percentile), respectively. The consumption is primarily accounted for by sausages, salami, pork flank (spiced and boiled) and ham. This consumption results in an exposure to NVNA of 33 and 90 ng kg bw(-1) day(-1) for adults and children, respectively. The exposure to VNA is significantly lower amounting to 0.34 and 1.1 ng kg bw(-1) day(-1) for adults and children, respectively. Based on a BMDL10 of 29 µg kg bw(-1) day(-1) a MOE value ≥17,000 was derived for the exposure to NA known to be carcinogenic (VNA including NSAR), indicating an exposure of low concern. The exposure to the NVNA is substantially higher and if found to be of toxicological significance the exposure may be of concern.

Pre-diagnostic acrylamide exposure and survival after breast cancer among postmenopausal Danish women

Acrylamide is a probable human carcinogen, with industrial contact, tobacco smoking and foods processed at high temperatures as the main routes of exposure. In animal studies oral intake of acrylamide has been related to cancer development, with indications that the increased cancer occurrence especially regards endocrine related tumors. In human epidemiological studies, dietary exposure to acrylamide has also been suggested related to higher risk of endocrine related tumors, like estrogen sensitive breast cancer. The aim of the present study was to evaluate if pre-diagnostic acrylamide exposure, measured by acrylamide and glycidamide hemoglobin adducts (AA-Hb and GA-Hb), were associated to mortality in breast cancer cases. Among 24,697 postmenopausal women included into a Danish cohort between 1993 and 1997, 420 developed breast cancer before 2001 and 110 died before 2009. AA-Hb and GA-Hb concentrations measured in blood samples were related to mortality by Cox proportional hazard models. Estimates are given per 25 pmol/g globin higher levels. Among non-smokers, higher concentrations of GA-Hb were associated to a higher hazard rate of breast cancer specific mortality (HR (95% CI): 1.63 (1.06-2.51)), the hazard rate among women diagnosed with estrogen receptor positive tumors was (HR (95% CI): 2.23 (1.38-3.61)). For AA-Hb the tendency was similar, but only statistically significant among those with estrogen receptor positive tumors (HR (95% CI): 1.31 (1.02-1.69)). In conclusion, the present study indicates that pre-diagnostic exposure to acrylamide may be related to mortality among breast cancer patients and that this may especially concern the most endocrine related type of breast cancer.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

Safety evaluation of plants collected from the wild served as food in Danish restaurants

DTU Orbit (03/08/2019) Safety evaluation of plants collected from the wild served as food in Danish restaurants Within the last decade the New Nordic Cuisine has received much media coverage. The restaurants have focused on increased use of locally grown plant food, including wild plants collected from the countryside. In addition, many cookbooks and guided nature walks have assisted interested consumers in the search for wild plants for culinary purposes. As part of a control campaign running from May–October 2016, the Danish food authorities investigated the use of plants picked from the wild, cultivated in private gardens or market gardens in restaurants and local food producers. Here we present examples of safety evaluations of some of the 50 plant species identified from this campaign based on published phytochemical investigations and toxicological data in humans. In the period from February to October, 2017, searches were performed in databases on bibliographic information using the preferred scientific name, and if relevant also synonyms. The full scientific papers were obtained if abstracts described ethnobotanical studies on food use in European countries prior to 15 May 1997 (the date the first novel food regulation came into force), constituents (especially if toxicological relevant), experimental laboratory animal studies on the toxicological effects of the plants, or cases of intoxications in humans or animals exposed to the individual plants. For the majority of the plants no or very limited phytochemical and safety information were available. Additionally, we found that of the 50 plants reviewed almost half contained compounds with toxic or potentially toxic effects if eaten. For many of the remaining plants, the data was insufficient to establish a safe edible amount. Many of the species may be considered novel food according to the EU regulation, since a food use to a significant degree in EU member states prior to 15 May 1997 could not be established. This review has demonstrated a strong need for better information on novel food status and safety of plants picked from the wild or plants previously mainly cultivated e.g. for ornamental use but now introduced as food, so that food producers, chefs and writers of cookbooks also in future have a stronger attention on whether the plants are safe to eat.

Are wild and cultivated flowers served in restaurants or sold by local producers in Denmark safe for the consumer

New Nordic Food has within the last decade received much media coverage with chefs of top restaurants using wild plants for foods. As part of a control campaign, the Danish Veterinary and Food Administration visited 150 restaurants and local food producers from May-October 2016 and investigated their use of plants picked from the wild, cultivated in private gardens or market gardens. Among the species used were the flowers from 23 plants. Here we present a safety evaluation of these flowers based on published phytochemical investigations and toxicological data in humans, farm animals, pets, or experimental animals. Of the 23 flowers reviewed, nine contained compounds with toxic or potentially toxic effects if eaten, two contained unidentified toxic compound(s), and four were flowers from plants with potentially toxic compounds present in other plant parts or related species. Many of the flowers may be considered novel, since a use to a significant degree in Europe prior to 15 May 1997 before Regulation (EC) 258/97 on novel food and novel food ingredients came into force could not be established. In conclusion, this review illuminates a striking lack of chemical and toxicological data of many of the proposed wild or cultivated flowers for food use.