Methods to Measure the Human Exposome
Martyn Smith
School of Public Health University of California, Berkeley http://superfund.berkeley.edu
Director, Berkeley Institute of the Environment and Superfund Research Program
The exposome changes paradigms for studying ‘environmental’ causes of disease
Traditional View
Exposome
Coverage Air and water pollutants All chemicals (exogenous and endogenous) from all non-genetic factors
Focus Population Individual
Study design Hypothesis testing (factor specific)
Hypothesis generating (discovery & omics) Allows for EWAS
Resolution Qualitative (self reports) Quantitative (measurements)
Monitoring External environment Internal environment
Time frame Mostly adult years Conception through early adulthood
Scientific framework
Reductionist Complex systems
Go beyond current hypotheses
BPA
PBDE
As
Benzene
Phthalates
PCBs
BADGE
DINCH
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Capturing all exposures
S.M. Rappaport and M.T. Smith, Science, 2010: 330:460-461
EXPOSURES ARE CHEMICALS and the blood exposome includes all chemicals in the body .
The microbiota: Comprise 90% of the cells and 95% of protein-coding genes in the human body
Measuring the Blood Exposome
Enable EWAS and generate biomarkers of exposure and
disease risk
0
10
20
30
40
50
60
70
80
90
100
1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05
Cum
ulat
ive
perc
ent
Blood concentration (µM)
DrugsFoodsPollutantsEndogenous
Venlafaxine
Aspirin
Simvastatin
Digoxin
Estradiol
Testosterone
Cortisol
Homocysteine
Cholesterol
Malondialdehyde Benzene
Lead
DDE Arsenic
PCB 170
Perfluorononanoic acid
Hexachlorocyclohexane
BDE 100
Cotinine
OCDD
Trichloromethane
Acetaldehyde
Folic acid, Vitamin D3
Sulforaphane
Trimethylamine-N-oxide
γ-Tocopherol
Ethanol
Solanidine
β-Carotene
Caffeine
Aflatoxin B1
Genistein
Normal blood concentrations
(1,561 chemicals)
1,000-fold
Rappaport S. et al., Environ Health Perspect, 2014
0
10
20
30
40
50
60
70
80
90
100
1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05
Cum
ulat
ive
perc
ent
Blood concentration (µM)
DrugsFoodsPollutantsEndogenous
Simvastatin
Digoxin
Estradiol
Testosterone
Benzene
DDE Arsenic
PCB 170
Perfluorononanoic acid
Hexachlorocyclohexane
BDE 100
Cotinine
OCDD
Trichloromethane
Folic acid, Vitamin D3
Solanidine
Aflatoxin B1
Genistein
Untargeted methods not currently suitable for serum concentrations < 0.1 µM
Rappaport S. et al., Environ Health Perspect, 2014
More than 30,000 small molecules detected in 0.1 ml of serum Less than 10% of the features are human metabolites
Siuzdak lab at Scripps
Million molecule in a minute exposome
A glass of Merlot has more than 6,500 distinct compounds. The average cup of coffee contains about 8,200 different natural chemicals.
From Susan Burch lab; http://www.ubc.ca/okanagan/chem/faculty/murch.html
Components of exposome - NOW • Electrophiles: Adductome of serum albumin • Metabolome: ~30,000 small molecules • Endocrine disruptors: ER and AR cell based assays • POPs: AhR cell based assay • Metals : ~20 easily measured • Infectious agents: Antibody array, subtractive sequencing • Stress: Telomere length, CD28, cortisol, amylase etc. • Oxidative stress markers: isoprostanes etc. (Panel) • Markers of inflammation: cytokines, C-reactive protein (Panel) • Early responsome signatures: transcriptome, methylome,
cellular immune response, etc.
Most measurements on serum or peripheral blood cells
Hormone Agonists and Antagonists
CALUX Luciferase Assay of Estrogenic and Androgenic Activity in Serum
Transfected cells plated into 96-well microplates
Plasma added to each well and incubated
Wells washed, cells lysed and luciferase activity measured
*Adjusted for BMI, number of live births, age at menarche, use of hormone replacement therapy, and family history of breast cancer.
Geometric means (95% CI) of serum biomarkers in postmenopausal breast cancer cases and controls, the Singapore Chinese Health Study
Biomarker Cases (n=169) Controls (n=426) Two-sided P*
Estrone (pM) 404.70 (355–461.36) 335.96 (308.28–366.12) 0.02
Estradiol (E2; pM) 66.26 (57.26–76.66) 58.82 (53.46–64.74) 0.19
Free E2 (pM) 1.38 (1.2–1.6)
1.16 (1.06–1.28) 0.05
ERa activity (pM E2 equivalent)
25.54 (24.66–26.44) 24.40 (23.84–24.96) 0.03
ERb activity (pM E2 equivalent)
25.22 (24.24–26.24) 24.22 (23.6–24.86) 0.10
Estrone and ERa-mediated bioactivity are related to increased breast cancer risk
V.V. Lim et al, Endocrine-Related Cancer (2014) 21, 263–273
Di(2-ethylhexyl) fumarate (DEHF) identified as EDC in bottled water by CALUX plus Hi-Res MS
Antiestrogenic (A) and antiandrogenic activity (B) of 18 bottled waters
By combining experimental and in silico MSn data compound identified as DEHF from 24,520 candidates Wagner M, et al (2013) PLoS ONE 8(8): e72472.
Human Serum Albumin (HSA)
• One free thiol (Cys34) • Represents 90% of free thiols in serum • Scavenges ROX and other electrophiles
• Preferred site for adduction of electrophiles in serum (conserved in all mammalian species)
Cys34
• Most abundant protein in serum
• 585 Amino acids • 35 Cys (-SH) residues
• 34 Used for disulfide bonds
Population A Population B
HSA
T3 Peptides
Adduct profiles
Sample ID
Add
ed m
ass
Sample ID
Add
ed m
ass
HSA-Cys34 adduct
Internal standard
Identify abundant and discordant adducts
LC-MS/MS
Digest
Cys34 Adductomics of Albumin
Rappaport SM, Li H, Grigoryan H, Funk WE, Williams ER. Adductomics: characterizing exposures to reactive electrophiles. Toxicol Lett. 2012;213(1):83-90.
Human Sequence
Subtraction • Non-Human
sequences detected by blasting against known databases of human sequence
Kostic, AD et al. “PathSeq: software to identify or discover microbes by deep sequencing of human tissue.” Nat. Biotech. 2011.
Non-Human Reads from PBMC RNA Subtraction Pathseq_Cloud
Total number of reads 84538500
Total number of reads after duplicate remover 84528666
Total number of unmapped reads after Maq 1 alignment (Database: MAQ1) 19716672
Total number of unmapped reads after Maq 4 alignment (Database: MAQ4) 4306112
Total number of unmapped reads after repeat masker 3066312
Total number of unmapped reads after Megablast (Database: BLAST1 & 2) 30201
Total number of unmapped reads after Blast N1/N2 (Database: BLAST1 & 2) 29212
Total number of unmapped reads 29212
Starting with 84.5 million reads...
29,212 do not map to the human genome.
Sample Total Reads Unmapped
1 53M 7,437
10 100M 22,541
17 120M 24,698
21 84.5M 29,212
Cell line 42M 7,872
Data from benzene study samples and a cell line
Akers, N. et al, unpublished data
Semenov et al, 2012
Distribution of small RNAs in Plasma vs. Cells
Vaz et al, 2010
Normal Plasma Normal PBMC
New Methods will be Needed • Improve mass spectrometry to detect >1 million
chemicals in human blood and other biofluids • Characterize and detect more adducts using MS • Develop other methods of detection • Use microfluidics and robotics to handle small
volumes • Engage bioengineers, chemists, biostatisticians and
computer scientists in project • International birth cohorts offer a great resource • Involve scientists from many countries (it took 6,000
physicists to discover Higgs boson)
Future of the exposome and disease etiology
• Transformative research happens once in a generation • Between 1988 and 2010 genomic research dominated
investigations of disease etiology despite disappointing results
• Exposomic research will find causes of disease and could dominate the next generation of etiologic research –This will require integrated omics technologies -
that measure chemicals comprehensively and efficiently - combined with advanced bioinformatics
Thanks to my whole lab team
Thanks
Major support from NIEHS through grants U54ES016115 and P42ES04705 and from the ACC Long-range Research Initiative
Stephen Rappaport Luoping Zhang Sylvia Sanchez Phum Tachachartvanich Christine Skibola Jacques Riby Sarah Daniels Fenna Sille Nicholas Akers Hasmik Grigoryan Laura Fejerman Sue Hankinson
