Methods and Issues in Using Biological Measurements in Epidemiologic Research: Nutritional Biomarkers and Disease Mechanisms

Transcription

1 EPI573, Autumn 2008 Methods and Issues in Using Biological Measurements in Epidemiologic Research: Nutritional Biomarkers and Disease Mechanisms I. Biotransformation Enzymes II. Peripheral Leukocyte Expression Arrays III. Gut Bacterial Profiling: Phytochemical Metabolizing Phenotypes

2 BIOMARKERS OF DIETARY INTAKE OR EXPOSURE Why? to provide biochemical data on nutritional status by generating objective evidence to provide objective evidence of a dietary pattern to validate dietary assessment instruments or self-reported data. to establish the biological link between the nutritional factor and a physiological or biochemical process, when the concentration of the micronutrient or dietary constituent is measured in a peripheral tissue.

3 Relationship between body compartments and specimens that can be assayed for dietary biomarkers Ingestion of food Respiratory Tract EXPIRED GASES SLOUGHED CELLS Tissue* GI Tract BLOOD Skin SWEAT Liver Kidney SALIVA HAIR FECES URINE * muscle, breast, prostate, etc.

4 FUNCTIONAL BIOMARKERS markers of physiologic, biochemical, or genetic effects of a nutrient or dietary constituent the first limiting biochemical system, e.g., a particular enzymatic pathway can be used to identify the dosage of a nutritional factor needed for a clinically meaningful response or to define optimum nutrient status ideal discrete functional marker reflects the direct effect of a dietary constituent - hard to do in intact humans rely primarily on a downstream marker

5 Functional Biomarkers of Dietary Exposures Susceptibility (e.g., genetic polymorphisms) Transcription DNA mrna Protein Protein Function e.g., enzyme activity Translation Substrate Product Down-stream Effects

6 Biotransformation Enzymes and Diet Do dietary constituents alter biotransformation enzyme activities and carcinogen metabolism?

7 Action of Biotransformation Enzymes Environmental exposures Drugs Endogenous compounds Plant Compounds BIOTRANSFORMATION Activation + Conjugation Activation Detoxified Compounds Excretion Reactive Intermediates DNA damage Cell toxicity

8 Biotransformation Enzymes and Cancer Processes Procarcinogen Ultimate carcinogen Excretion DNA adducts Phase I activating enzymes - P450s, etc Phase II conjugating enzymes - GSTs, etc Somatic alterations of genes Abnormal DNA and cell replication Precancerous lesions/dysplasia Cancer Metastasis

9 Biotransformation Enzymes and Cancer Processes Procarcinogen Ultimate carcinogen DNA adducts Somatic alterations of genes Abnormal DNA and cell replication Precancerous lesions / dysplasia Cancer Metastasis Steroid hormones, etc Excretion P450 activation Intermediates Conjugation

10 Approaches to Measuring Biotransformation Enzymes In Vivo Enzyme activity and expression in various tissues Metabolites of: endogenous compounds environmental agents Probe Drugs caffeine - CYP1A2, NAT2, xanthine oxidase chlorzoxazone - CYP2E1 debrizoquine - CYP2D6 acetaminophen/paracetamol - UGTs, STs

11 Measurement of mrna and Protein: Chargrilled Meat Induces Expression of Enterocyte CYP1A1 N = 10 5-day washout and 6-day chargrilled meat Duodenal and rectal biopsies: days 5 and 12 Fontana et al, Gastroenterol, 1999

12 Enterocyte CYP1A1 Protein Correlates with White Blood Cell PAH-DNA Adducts Day 11 Day 26 P = 0.05 Fontana et al, Gastroenterol, 1999

13 Measurement of Enzyme Protein: Effect of Brussels Sprout Consumption on ng/ml Plasma GST-α Females n=5 Males n=5 * Cross-over 300 g Brussels sprouts 1 week Basal Sprouts Nijhoff et al., Carcinogenesis, 1995

14 Measurement of Enzyme Activity: Effect of Broccoli on Salivary GST Activity miu/ml saliva Quit Coffee Quit Broccoli N=1 300 g broccoli days Start Broccoli Day Sreerama et al, 1995

15 WBC GSTμ Activity with Vegetable Diets: Responses in GSTM1+ and GSTM1-null pmol/mg/min 350 GSTM1 + GSTM1 null Basal Brassica Allium Apiaceous Adjusted LS-means + SE

16 Brassica and Allium Vegetables Increase WBC GST-μ Activity in GSTM1+ Women change pmol/mg/min * * Women (n = 7) Men (n = 11) Basal Brassica Allium Apiaceous Adjusted LS-means + SE

17 Measurement of Endogenous Compounds: Indole-3-Carbinol Induces Estrogen 2-Hydroxylation 2OHE1:E I-3-C Cellulose Placebo Months of treatment 20 women/group 3-mo treatment 400 mg I-3-C 20 g α-cellulose placebo urinary ratio of 2OHE1:E3 Bradlow et al, CEBP 1994

18 Measurement of Exogenous Compounds: Watercress Increases Excretion of Gluc-Conjugates of Tobacco-Smoke Metabolites % change 60 NNAL+NNAL-Gluc * Watercress trans-3'-oh cotinine 12 smokers 57 g watercress 3-day treatment Hecht et al, CEBP, 1995, 1999

19 URINE CAFFEINE TEST CYP1A2, NAT2, xanthine oxidase Administered on morning of Day 8, after a 12-h, overnight fast 200 mg caffeine (NoDoz) 240 ml water light breakfast (120 ml orange juice and muffin) Urine collected during fifth hour Caffeine metabolites analyzed by HPLC (Butler et al., Pharmacogenetics, 2:116, 1992)

20 Major Pathway of Caffeine Metabolism in Humans NAT2 AFMU 5-acetylamino-6-formylamino- 3-methyluracil AAMU 5-acetylamino-6-amino- 3-methyluracil CAFFEINE 137X (1,3,7 trimethylxanthine) PARAXANTHINE 17X (1,7 dimethylxanthine Z CYP1A2 CYP1A2 1X 1-methylxanthine 1U 1-methylurate xanthine oxidase

21 Measurement of Caffeine Metabolites HPLC analysis Ratios: 1U + 1X + AFMU 17U 17X + 17U 137X 1U + 1X + AFMU 17X 17X 137X

22 Pan-Fried Meat, High in Heterocyclic Aromatic Amines, Increases CYP1A2 Activity Caffeine metabolite ratio * N = 66 nonsmokers 1-week treatment Pan-fried meat 100 C 250 C 0 Low-temp High-temp Sinha et al, Cancer Res, 1994

23 Brassica Increase and Apiaceous Vegetables Decrease CYP1A2 Activity Caffeine metabolite ratio 10 P = P = Basal Brassica Allium Apiaceous Adjusted LS-means + SE Lampe et al, Carcinogenesis, 2000

24 Down-stream Effects Functional Biomarkers of Dietary Exposures: Profiling Susceptibility (e.g., genetic variation) Epi/genomics Transcription Translation DNA mrna Protein Protein Function e.g., enzyme activity Transcriptomics Proteomics Substrate Product Metabolomics

25 Functional Biomarkers of Environmental Exposure Provide a measure of exposure Reflect the degree of physiologic and biochemical response to the exposure Peripheral leukocytes see only some exposures and have a limited repertoire of responses, but often exposed similarly to target tissues

26 Biologic Sample Processing How stable are the measures of interest? How sensitive are they to light? Temperature? What time-frame do you have for processing? -- How quickly do the markers degrade?

27 Sample Handling: Peripheral Leukocyte Gene Expression Over Time 0 vs 1 h 0 vs 3 h 0 vs 6 h 0 vs 24 h

28 Use of Peripheral Blood Leukocyte Gene Expression to Monitor Tobacco Smoke Exposure Reasoning: We could verify smoking exposure by both self-report and the measurement of plasma cotinine concentrations If tobacco smoking did not yield a detectable and repeatable signature, other more subtle environmental exposures were even less likely to do so If we could detect a signature for tobacco, other exposures, behaviors, and characteristics could also be explored for characteristic signatures.

29 Sample and Data Collection Two blood draws, one week apart. Blood samples were collected in the morning, between 7 and 10 AM, after a 10-hour fast. Data on demographics, smoking history, diet and exercise obtained using VITAL questionnaire.

30 Smokers vs Nonsmokers: Microarray Data Analysis Training Set: Gene-expression data from 65 individuals (32 smokers and 33 nonsmokers) to select and optimize a set of reporter genes. Test Sets: Array data for 20 other participants (10 smokers and 10 nonsmokers, equally distributed by sex; TEST1) and follow-up visit 2 samples for 17 of these 20 individuals as a separate test set (TEST2).

31 Expression Data Matrix for Training Set 36 Optimal Reporter Genes for Cotinine Exposure Contig 57903RC BRD3 AI205537RC Contig 38824RC FLJ10254 TNNT1 CYP1B1 NRG1 HCA112 KIAA1024 FLJ20701 IL1B DNAJC7 Contig 35145RC Contig C1QB LR8 EPB41L3 USP6 CCR2 PFKFB3 Contig 38043RC CSPG2 NRG1 NRG1 HML2 Contig 65401RC AOC2 Contig 23408RC FLJ20202 TPST1 IDS LOC51107 RPGRIP1 NESG1 PBX2 a b Smokers: cotinine >30 ng/ml 36 reporters 27 positively assoc 9 negatively assoc 26 characterized genes 10 ESTs, partial cdnas, and hypothetical proteins. Nonsmokers: cotinine 0 ng/ml Participants

32 Misclassification of Individuals in Training Set Plasma cotinine ng/ml Smokers misclassified as nonsmokers Nonsmokers misclassified as smokers

33 Expression Data Matrices across 36 Optimal Reporter Genes for Cotinine Exposure Tests 1 and 2 T T2 c

34 INTERVENTION: Lymphocyte Gene Expression Differentially Induced in Equol-Producing and Nonproducing Women 30 postmenopausal women ~900 mg isoflavones for 84 d Gene expression array of peripheral lymphocytes 27 genes changed with isoflavones Stronger effect on estrogenresponsive genes in equol producers than nonproducers. Niculescu et al, J Nutr Biochem 18:380, 2007.

35 Intestinal Microbiota and Impact on Health ~ 100 trillion microorganisms (10 times more than cells in our body) Dominated by relatively few divisions (e.g. Proteobacteria, Cytophage, Flavobacterium, Bacteroidetes, Firmicutes) but highly diverse at the strain/species level at least 800 species of bacteria, mostly unknown) Another genome buried in our body Microbiome Individual Specific Dynamic Backhed et al., Science 307: 1915

36 Phytochemical Metabolism by Gut Bacteria Deconjugation of glycosides, glucuronides, and sulfates Ring-cleavage, demethylation and dehydroxylation reactions Some bacterial metabolites more bioactive than parent compounds

37 Metabolism of Phytochemicals In Vivo Hydrolysis of glycosides Metabolism and absorption of aglycones Conjugation in intestinal epithelium and liver Excretion Enterohepatic circulation Adapted from Setchell, 1995

38 Hop Prenylflavonoid Metabolism HO OMe OH O OH Xanthohumol (X) Michael addition HO OMe O O Isoxanthohumol (IX) OH Bacterial O-demethylation HO OH OH O OH Desmethyl-xanthohumol Michael addition HO 6 8 OH 8-Prenylnaringenin HOPEIN (8-PN) O 1 O 2 OH OH HO O + OH O 300x 6-Prenylnaringenin more estrogenic than genistein Slide courtesy of Sam Possemiers, Ghent University

39 Isoxanthohumol Conversion to 8-prenylnaringenin by Fecal Cultures from 12 Individuals Concentration (mg/l) IX 8-PN 0 * * * * * * * * A B C D E F G H I J K L Possemiers et al., J Ag Food Chem, 2005 * = not detected Slide courtesy of Sam Possemiers, Ghent University

40 Bacterial Production of Equol and ODMA HO O HO O O Daidzein OH O Dihydrodaidzein OH HO O 80-90% of individuals produce HO OH O OH O-Desmethylangolensin HO OH O Equol OH Cis/Trans-isoflavan-4-ol OH 30-50% of individuals produce

41 Urinary Equol Excretion with Soy Challenge nmol/d Equol Excreters Equol Non-excreters 1 Subject 1 equol 60 Lampe et al., PSEBM 217: , 1998

42 Equol-Producing Capacity and Human Health Inversely associated with circulating estrogens and androgens, and positively correlated with SHBG. Adlercreutz et al, J Steroid Biochem 27: , Positively associated with 2-OH/16αOHE1 ratios in premenopausal women. Atkinson et al, J Steroid Biochem Mol Biol 86:71, 2003 Inversely associated with prostate cancer risk in Japanese men. Akaza et al., Jpn J Clin Oncol 32:296, 2002.

43 Phenotyping for Equol- and ODMA-Production in Soy Non-Consuming Populations Standardized system to determine phenotype Involves a 3-day soy (daidzein) challenge and a morning first-void urine collection analyzed for daidzein, equol, and ODMA Minimum burden to individuals Can be used in large, population-based, epidemiologic studies

44 Equol and ODMA Phenotyping Kit 3 servings of soy >10 mg daidzein Urine collection kit and mailer Instructions

45 Urinary Isoflavone Stability at 25 C for 14 Days Urine collected from known equol-producers Aliquotted and stored at ~25 C for up to 14 days before freezing at -20 C Analyzed for daidzein, equol, and ODMA by GC- MS Daidzein Equol ODMA ng/ml ng/ml ng/ml Frankenfeld et al, Exp Biol Med 229: ,

46 Predicted Prevalence of Equol- and ODMA- Producers in Relation to Age 1.9 O-DMA-producer phenotype Prevalence of producers Equol-producer phenotype P=0.01 P= n=410 Age in years

47 Is the phenotype stable? Evaluated within-individual concordance of equol-producer and ODMA-producer phenotype measured at two time points one to three years apart. Recruited individuals who had previously participated in The Family Study of Soy Digestion > 10 years of age No allergy to soy Phenotyped 112 individuals for equol- and ODMA-production > 44 ng/ml in 4 ml urine Documented oral or IV antibiotic use within 3 months

48 Concordance of Equol Phenotypes (%) at Time 1 and Time 2 Time 2 Time 1 Equol Producer Equol Producer 34 Equol Nonproducer Equol Nonproducer Observed agreement = 82% Agreement expected by chance = 51.2% κ = 0.64 (SE: 0.10) Frankenfeld et al., Br J Nutr in press, 2005.

49 Does the prevalence of daidzein-metabolizing phenotypes differ between populations that are low- and high-soy consumers? Recruited Korean American women and girls in the Seattle area > 10 years of age No allergy to soy No antibiotics within 3 months of participation Phenotyped for equol- and ODMA-production Compared phenotype prevalence in the Korean Americans with those of Caucasian women and girls in The Family Study of Soy Digestion

50 Equol-Producer Phenotype Prevalence Higher (p=0.015) in Korean Americans Compared to Caucasians Age % of age quartile Korean American Caucasian n=90 n= * * Age quartiles based on Korean-American women Song, et al 2006.

51 Can we distinguish individuals with different daidzein-metabolizing phenotypes using fecal microbial community profiles? Objective To compare, using terminal restriction fragment length polymorphism (trflp), the fecal bacterial community profiles that develop when feces from different individuals are incubated with daidzein.

52 Terminal Restriction Fragment Length Polymorphism (trflp) Fecal Sample Isolation of Total DNA of Bacterial Cells Amplification of 16S rdna by PCR Restriction Enzyme Digestion (Hae III) and Cleanup Electrophoresis Analysis of trflp pattern

53 Method Isolate total bacterial DNA PCR-amplify 16S rrna gene using bacterial domain primers 27F- FAM and 1492R Digest with restriction enzyme - Alu I Y Run samples by capillary electrophoresis Analyze trflp patterns using internal standards of known base-pair size Time (s)

54 Multivariate analysis of trflp patterns derived from 16S rrna gene sequences amplified from intestinal microbial communities n=25 Alu I Axis bp. 112 bp, 129 bp, 150 bp Axis 2 (28%) 211 bp 1.5 Equol Axis 2 (28%) 37 bp Axis 1 ( 65%) 236 bp ODMA DHD DHD + Equol DHD + ODMA Hullar et al, unpublished

55 Phytochemicals Metabolism: Summary Intake Exposure Interindividual difference in phytochemical metabolism and disposition may be affected by: Gut microbiota identity and activity Environmental exposures that influence gut microbiota