ERN3120 Part 1 March :00 13:00
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1 ERN3120 Part 1 March :00 13:00 Answer all questions. Read each question carefully before starting to answer it. You may write your answers in either English or Norwegian. Section A is in the form of multiple choice questions: you should indicate what you think is a correct statement by ticking the box. Spend no more than one hour on this section. Section B consists of 5 questions requiring written answers. Spend two hours on this section. Section C requires you to answer questions based on reading a scientific paper. Spend up to one hour on this section. In Sections B and C, start each answer on a new page. Because some questions require marking on the question sheet, you must return the question sheet marked with your candidate number, together with the written answers. 1
2 Section A. Multiple choice questions. Spend about one hour on this section. Mark the box beside those statements that you think are correct. There may be one or more correct statements. A.1. DNA damage Oxidised bases in DNA are frequently measured as an index of oxidative stress, which may be involved in various chronic human diseases. The number of 8 oxoguanines in the DNA of a normal human cell is estimated to be approximately one per million guanines. (Assume that the DNA content of a cell is 2x10 12 Daltons and the molecular weight of a nucleotide in DNA is 333 Daltons.) This level of damage corresponds, approximately, to how many 8 oxoguanines per cell? Choose one of the following: true A.2. Antioxidant supplements Many people take antioxidant supplements because they believe these will protect them against cancer and heart disease. Subject A, a non smoker, takes a high dose β carotene capsule every day. Subject B, also a nonsmoker, takes high doses of vitamins C and E. Which statement(s) do you agree with? Subject A has a lower chance of contracting lung cancer than does subject B. false Subjects A and B will be protected against heart disease by the supplementation. false Subject A may find her skin becoming slightly yellow. true Both subjects would be better off taking a placebo pill. true It is impossible to predict any disease outcome from these facts. true A.3. DNA damage, DNA repair and cancer Which statement(s) do you agree with? The various DNA repair pathways start with the removal of damage by specific enzymes; the later steps of gap filling and ligation are identical for all pathways. false In most cell types, DNA repair activity is absent: it is switched on by the presence of DNA damage. false 2
3 Aflatoxin is a contaminant of grains and seeds stored in damp conditions. It is believed to cause colon cancer after activation by phase I (P450) enzymes. false it is linked to liver cancer In people with a defective allele of a mismatch repair gene, there is a strong possibility of a mutation occurring in the other allele, resulting in hereditary non polyposis colon cancer (HNPCC). true Oxidation by free radicals is believed to be a major cause of DNA base damage in humans, and 8 oxoguanine is one of the most often measured oxidation products. true A.4. Lipid oxidation This graph (blue line) depicts the oxidation of LDL (low density lipoprotein) by a free radicalgenerating system. It also shows the consumption of the antioxidants that are present in the LDL particles, in terms of the percentage of the initial level remaining at different times of incubation. What conclusions can be drawn from this graph? Antioxidants are inactivated by the scavenging of free radicals true Oxidation only occurs when the total antioxidant level is low true Vitamin E delays the consumption of the carotenoids (lycopene and β carotene) false The concentration of β carotene in LDL at the beginning was lower than the concentration of lycopene. false A.5. Vitamin E Which statement(s) do you agree with? γ Tocopherol is preferentially retained in the body false Tocopherol transport protein is present in the plasma false Vitamin E has 8 isomers true 3
4 Vitamin E, like vitamin A, is stored in the liver false A.6. Vitamin A deficiency Vitamin A deficiency may lead to: Activation of natural killer (NK) cells false Decrease in Th2 stimulated antibody production true Depression of IgG antibody response true Increased risk of infection false no difference in risk, but infections more severe A.7. Which of these statement(s) about the vitamin D receptor (VDR) are true? It is a nuclear receptor true After its activation, biological effects can be observed after 2 minutes false Activated VDR directly contacts the RNA polymerase II false It can both downregulate or upregulate transcription of its target genes true It requires the same dimer partner as the vitamin A receptor true Expression of the 1alpha hydroxylase gene is repressed by the VDR true A.8. Iodine Which statement(s) do you agree with? Most of the iodine taken in by the average adult Norwegian comes from dairy products. true The recommended daily intake of iodine, 150 micrograms, applies to adults and children, though in pregnancy and while breast feeding more than this should be taken. true 25% of the population in Norway takes in less than the recommended level of iodine and is therefore likely to suffer from goitre. false Fish is generally a good source of iodine true Iodine deficiency during pregnancy can cause severe mental deficiency in the child. true A.9. Essential minerals and trace elements Essential minerals are elements with recommended intake of >100 mg/day. Trace elements have recommended intake of <100 mg/day. Ultra trace elements are minerals with established, estimated or suspected daily requirement of <1mg. Which of these statement(s) do you agree with? There is no good evidence that fluorine is essential, though it is classed as an ultra trace element. true Selenium has higher toxicity than arsenic true Iron is a trace element true 4
5 Many enzymes involved in protection against oxidative stress are dependent on trace elements. true Fruits and vegetables are poor sources of essential minerals and trace elements, and vegetarians are likely to be deficient. false A.10. General principles of food toxicology Which statement(s) do you agree with? A dose response curve is used to establish the threshold for a toxin s effect. true The threshold dose can be calculated from LD50. false The acceptable daily intake (ADI) of a substance is based on the lowest observed effect level (LOAEL). false The acceptable daily intake (ADI) of a substance is based on the no observed adverse effect level (NOAEL). true A.11. Toxins in food There are four classes of toxins found in food: Naturally occurring toxins Environmental contaminants, biological and chemical Food additives Derived toxic substances in food Which statement(s) do you agree with? Over the years, various food additives have been banned because they have been associated with cancer or other diseases in animals. true If a toxic compound is highly bioavailable, it means it will be taken up with lipids and accumulate in adipose tissue. false Favism is an allergic reaction to fava beans (Vicia faba) common in people of Mediterranean origin. false (not an allergy) Nitrosamines are derived substances, produced by reaction of nitrites with secondary amines in conditions of low ph such as the stomach. true Because nitrites can cause DNA damage and mutations, their use as a preservative is now banned. false A.12. Cancer epidemiology (a) Which of these statements do you consider to be correct? Breast feeding protects against breast cancer. true Breast feeding protects against cervical cancer. false There is no convincing evidence that dietary factors modify the risk of cervical cancer. true It is unlikely that dietary factors modify the risk of lung cancer. false 5
6 The risk of colorectal cancer is almost certainly increased by high consumption of processed meat. true A.13. Cancer epidemiology (b) Which of the following are possible reasons for a rise in age standardised cancer incidence rates? Existence of screening programmes true Increasing population size false An increase in prevalence of risk factors true Improved survival rates false A.14. Cancer in the developing world Which 4 of these cancers are the most common in the developing world? Melanoma Colon Stomach yes Prostate Liver yes Lung yes Ovary Oesophagus yes 6
7 Section B: Spend about two hours on this section. B.1. Flavonoids Quercetin a toxic phytochemical? Read these notes on two experiments, and then answer the four questions. Cell culture experiment: cytotoxicity of quercetin Cytotoxicity of quercetin Relative colony number (%) Quercetin concentration (μg/ml) Cultured human cells were treated for 30 minutes with quercetin, a common flavonoid, and then placed in growth medium in a culture dish and incubated at 37 C. The concentration of cells was low, so that each surviving cell could attach to the bottom of the dish and divide, forming separate colonies that after one week were big enough to be counted by eye. The graph shows the number of colonies present after treatment with different quercetin concentrations, expressed as a percentage relative to the number of colonies in control, untreated cultures (100%). (i) In fewer than 100 words, what do you conclude from these experimental results? Quercetin is cytotoxic, ie prevents them from forming colonies, at concentrations above 20 µg/ml. This could be because cells are killed, or because they are prevented from attaching to the surface, or prevented from dividing. Human intervention study: In a human supplementation trial, a capsule containing 20 mg of quercetin was given to volunteers on one occasion, and a placebo capsule was given on another occasion. Blood samples were taken at 4 hourly intervals over a period of 24 hours afterwards. Quercetin (as a glycoside) was detected in the plasma at 4 hours and 8 hours after taking the capsule. Lymphocytes isolated from the 8 hour blood sample were treated with H 2 O 2 and DNA breaks were measured with the comet assay. The mean levels of DNA breaks (arbitrary units) were: after placebo, 344 units: after quercetin, 280 units. This difference was statistically significant. (ii) In fewer than 100 words, what do you understand from the result of the H 2 O 2 experiment? The lymphocytes from the volunteers taking supplement are more resistant to the damaging effect of 7
8 H 2 O 2. This indicates that they have a higher antioxidant capacity, and implies that in spite of the ability to cause damage quercetin in vivo acts in a protective way. (iii) Estimate, by making reasonable assumptions about bioavailability, blood volume, etc., what the concentration of quercetin in the plasma might be. Which of these concentrations is most likely? 100 ng/ml, or 50 µg/ml, or 25 mg/ml? Show your reasoning. Assume uptake of 20%, metabolism of half of this, leaving 10% or 2 mg distributed in a blood/tissue volume of, say, 10 litres; so concentration would be about 0.2 mg/litre, or 0.2 µg/ml, or 200 ng/ml. So 100 ng/ml is most likely. (iv) Would you regard quercetin as a harmful compound on the basis of the cell culture experiment, or as a beneficial agent on the basis of the human trial? Give reasons for your answer. Would you recommend healthy people to avoid eating foods containing quercetin? It is very unlikely that a concentration of 20 µg/ml would be reached in the body, so it is unlikely that any damage would be caused. On the other hand, protection can occur at a concentration of less than 1 µg/ml (see answer to (iii)) which might be achieved by consuming foods containing quercetin. So there is no reason to stop eating foods containing quercetin. B.2. Water soluble vitamins For each of these vitamins vitamin C, cobalamin (vitamin B12), thiamine (vitamin B1) list the following: a) main sources in the food b) cellular function (if several, list some main functions) c) name(s) of syndrome(s) or condition(s) that arise in deficiency d) symptoms and findings that are associated with deficiency of the vitamin e) describe briefly the interaction, if any, of alcohol with this vitamin Example of response: Vitamin B3 (niacin) a Sources of niacin include a wide variety of foods, such as meat(fish) products, egg, dairy, many vegetables, nut and whole grains. It is also synthesized from tryptophan, which is found in meat, dairy and eggs. (A bit beside the question: Niacin deficiency is therefore rare, usually found in populations eating mainly corn as staple. Native populations brought out the niacin in corn during coking by alkali (lime)). b Enzymes in energy-yielding metabolism, e.g., oxidative decarboxylation of pyruvate and alpha-ketoglutarate c d death Pellagra. Pellagra is classically described by "the four D's": diarrhea, dermatitis, dementia and e) Alcohol increases the risk of pellagra, and niacin deficiency increases the damaging effects of alcohol, including alcoholic dementia. 8
9 B.3. Genetics (i) Explain, and clearly distinguish, the terms multifactorial disease and complex polygenic disease. Multifactorial disease describes a disease with more than one contributory factor in its etiology. These factors can be genetic and/or environmental. A complex polygenic disease is a disease with several genes involved in its causation: it could also (but need not) have environmental causes too. Extra marks if examples given. (ii) Why might people with the same genotype show different phenotypes? Phenotype depends on genotype, but is also influenced by environment (which includes nutrition and lifestyle). (iii) Why are some people predisposed to becoming obese? What other factors are important in the etiology of this disease? Genetic factors are important in determining susceptibility. Children of obese parents are 10x more likely to be obese than children of parents with normal weight (but some of this effect is likely to be due to common environment, eg nutrition and lifestyle). Genetic factors responsible for predisposition: control of food intake, low basal metabolic rate, adipocyte differentiation. Many polymorphisms identified. Other factors: lifestyle, lack of exercise, psychological factors, tradition/culture. B.4. Calcium homeostasis A low concentration of calcium in the plasma triggers a series of metabolic events. Describe the main sequence of events, explaining how they lead to a restoration of the calcium level in plasma, (i) assuming that the supply of vitamin D and vitamin K is adequate and that liver function is normal. Fasit: PTH secretion from the parathyroid gland is enhanced, stimulating release of calcium from the bones. PTH also up regulates 1 alpha hydroxylase in the kidneys, thus activating 25 OH vitamin D to calcitriol, which in turn increases the uptake of calcium from the intestine. When the plasma calcium level is restored, PTH secretion stops. Mentioning of the effect of calcitonin is not warranted here. (ii) If, however, vitamin K status is low, describe how the patient's bone mass is gradually lost. Fasit: Vitamin K promotes the carboxylation of osteocalcin, a protein necessary for the calcification of bone matrix proteins. Furthermore, vitamin K is a ligand for the nuclear receptor SXR, stimulating the transcription of genes encoding several bone matrix proteins and osteoprotegerin (OPG), Hence, low vitamin K status reduces the steady state level of bone matrix proteins, and enhances the degradation of bone through activation of osteoclasts (RANKL/OPG ratio is increased). 9
10 B.5. Nanoparticles List the various ways in which nanoparticles can get into food. Why are there safety concerns about nanoparticles? Leakage from packaging; = 1 contamination from coatings on kitchen equipment; = 1 nanocapsules containing additives in functional foods; = 1 nanoparticles added to foods to prevent spoilage; = 1 nanoparticle sensors (monitoring edibility). = 1 Altogether 5 points Why are there safety concerns about nanoparticles? Because little is known about how nanoparticles behave in the body. They can enter cells and even the nucleus. They have properties that differ from the normal substance, on account of their large surface area/mass ratio (increasing reactivity). NPs are more chemically reactive than larger particles NPs have greater access to our bodies than larger particles Greater bioavailability and bioactivity may introduce new toxicity risks NPs can compromise our immune system response NPs may have longer term pathological effects We don t know What levels of nano exposure we are currently facing What levels of exposure could harm our health or if there is any safe level of exposure Altogether 5 points, depending how detailed is description of safety (in bulletpoints above) 10
11 Section C (one hour). C.1. Read the Abstract and Introduction in the article Resveratrol prevents epigenetic silencing of BRCA1 by the aromatic hydrocarbon receptor in human breast cancer cells. Place the correct name on the different components in this illustration, based on the information provided in the article. Activation of expression: ERα Ac Ac BRCA 1 promoter BRCA 1 gene DNMT1 Me (methylation) Ac (acetylation) ERα TCDD AhR Estrogen XRE RNA Double strand break H3 (histone 3) H4 (histone 4) Repression of expression: TCDD Me C.2. Explain in about 4 sentences with your own words what the main findings in the article are. C.3. What is the specific question the authors want to answer by doing the experiment described in fig. 2A and how do you interpret the results? C.4. Explain how phytochemicals can play a role in xenobiotic metabolism, and in particular in the metabolism of (pro)carcinogens. Note: It is not necessary to study the whole article. Most of the information you need is in the Abstract and Introduction. 11
12 Answers: 1. Activation of expression: Estro gen ERα mrna H3 H4 Ac Ac BRCA 1 promoter BRCA 1 gene Repression of expression: Ac TCDD AhR XRE DNMT1 H3 H4 Me BRCA 1 promoter BRCA 1 gene Double strand break 2. When the aryl hydrocarbon receptor is activated by the carcinogen TCDD, it prevents estrogen and estrogen receptor from transcribing the tumor suppressor BRCA1. The effect is caused by epigenetic modifications of nucleosomes at the BRCA1 promoter. The result of the inhibition is accumulation of double DNA strand breaks. BRCA1 repression and DNA double strand breaks can be prevented by pretreament of cells with the phytoestrogen resveratrol. 3. TCDD increases binding to the promoter, an increase that is partially prevented by estrogen, suggesting a competition by estrogen receptor and aryl hydrocarbon receptor. When cells are treated with resveratrol, binding of AhR to the promoter is reduced, showing a dose response relationship. 4. Among the more than 8000 different phytochemicals found in food plants there are many that are foreign to mammalian cells and many of these compounds can bind to the aryl hydrocarbon receptor. This receptor binds xenobiotic responsive elements in gene promoters and is responsible for transcription of phase I enzymes (cytochrome P450 enzymes). These enzymes can inactivate cancer causing substances (carcinogens), but can also activate substances, making them more carcinogenic (activating procarcinogens). Phytochemicals can thus regulate the amounts and 12
13 activities of phase I enzymes and thereby influence how cells at a later time point metabolize carcinogenic substances. 13
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