novel biomarkers for the risk assessment of exposure to arsenic in drinking water
TRANSCRIPT
Jenna Currier, Ph.D.
ORISE Postdoctoral Candidate Visit
Novel biomarkers for the risk assessment of exposure to
arsenic in drinking water
October 28, 2013Research Mentor: Miroslav Styblo
2 of 43
Overview
Introduction Hypothesis Results
Method optimization and validation Population-based study
Conclusions Questions
3 of 43
Arsenic (As): A global public health issue
Inorganic arsenic (iAs) is a naturally occurring carcinogenic metalloid found in water sources worldwide
Common oxidation states in aquifers: +3, +5
Tens of millions are exposed to this toxic element
Current EPA/WHO limit: 10 ppb
(Photo: Richard Wilson, Harvard University)
Cancer of the skin, bladder, lungs and liver
Skin lesions Diabetes Hypertension, cardiac arrhythmias Peripheral neuropathy, black foot disease
Adverse Effects of Chronic As Exposure
4 of 43
A global (and local) public health issue
Ryker, S.J., Nov. (2001) Mapping arsenic in groundwater, Geotimes. 46: 34-36.
Carolina Slate Belt
Dr. Avner VengoshDuke University
5 of 43
Arsenic biomethylation
D. Thomas, et al. (2007) Experimental Biology and
Medicine 232:3-13.
Arsenic (+3 oxidation state) methyltransferase (AS3MT) mediates the metabolism of iAs AS3MT is expressed in many tissues, including liver As methylation facilitates excretion
Methylarsonite (MAsIII) and dimethylarsinite (DMAsIII) are more biologically active than iAs and their pentavalent counterparts Free MAsIII and DMAsIII are unstable in the presence of
O2 (e.g., in urine)
6 of 43
iAs-induced diabetes: in vitro evidence
Diabetes
Arsenic
AS3MT ?
1. Trouba, K. J., et al., (2000) Toxicol. Appl. Pharmacol. 168, 25-35.2. Steffens, A. A., et al., (2011) Toxicol. Appl. Pharmacol. 250, 154-161.3. Walton, F.S., et al., (2004) Toxicol. Appl. Pharmacol. 198, 424-433.4. Paul, D., et al., (2007) Environ. Health Perspect. 115, 734-742.5. Douillet, C., et al., (2013) Environ. Health Perspect. 119(8): 1104-1109
o Peripheral tissues o iAsIII inhibits adipocyte1 and myoblast2 differentiationo Subtoxic concentrations of AsIII species decrease
glucose uptake in cultured adipocytes3
o MAsIII > DMAsIII >> iAsIII
o AsIII species decrease GLUT4 translocation to plasma membrane in cultured adipocytes3,4
o iAsIII and MAsIII inhibit phosphorylation of PKB4
o Pancreaso AsIII species decrease pancreatic β-cell insulin secretiono MAsIII, DMAsIII > iAsIII
o Likely mediated by inhibition of insulin transport vesicle packaging or translocation5
7 of 43
iAs-induced diabetes: in vivo evidence
Diabetes
Arsenic
AS3MT ?
1. Maull, E.A, et al., (2012) Environ. Health Perspect. NTP Workshop Report 120:1658-70.
2. Paul, D., et al., (2007) Toxicol. Appl. Pharmacol. 222: 305-314.3. Paul, D., et al., (2011) Environ. Health Perspect. 119(8): 1104-1109.
o Diabetic mouse modelo 8 week exposure to 50 ppm As in drinking water
resulted in impaired glucose tolerance2
o iAs exposure and diet-induced obesity produced a unique diabetic phenotype characterized by impaired glucose tolerance in the absence of fasting hyperinsulinemia3
o Diabetes mellituso Increased prevalence of diabetes in populations
exposed to high levels (≥150 ppb) of iAs in drinking water1
8 of 43
Rationale
Quantification of AsIII species in biological systems must be performed to determine the effects of trivalent arsenicals on pathways critical for glucose homeostasis and the development of diabetes.
9 of 43
Quantifying AsIII and AsV species
Gaseous arsines and methyl-substituted arsines are generated directly from the sample
No extractions or pretreatments are needed for analysis of AsIII species
Sample volumes of 0.5 mL Detection limits range from 9 to 20 pg Seven As species can be quantified using a two aliquot
approach
Hydride Generation - Cryotrapping -Atomic Absorption Spectrometry (HG-CT-AAS)
The HG-CT unit can be connected to an ICP-MS for detection limits ranging from 40 fg to 2 pg
10 of 43
Method Principles
iAsIII
MAsIII
DMAsIII
TMAsVO
iAsIII+V
MAsIII+V
DMAsIII+V
ArsineAsH3
Methyl-Arsine
CH3AsH2
Dimethyl-Arsine
(CH3)2AsH
Trimethyl-Arsine
(CH3)3As
pH 6 Tris buffer + strong reductant
(NaBH4)
pH 6 Tris buffer + strong reductant
(NaBH4)
Cryotrapping
Sample divided
Direct analysis
2% cysteine pre-reduction
-62.5°C
1.2°C
35.6°C
53.8°C
11 of 43
As speciation in urine
In urine, DMAsIII can be completely oxidized in less than 1 day.
0 5 10 15 20 250
20
40
60
80
100 Urine 1 dry iceUrine 1 ice
Storage Time (hours)D
MA
sIII
(% o
f Con
trol)
15 ppb DMAsIII spiked into a control urine sample and stored on dry ice or ice
for up to 24 hours.
Del Razo, L.M., et al. (2011) Environ. Health, 10: 73-84.
As metabolite profiles in urine reflect
excretion and not necessarily target tissue retention
12 of 43
AsIII species may be stable incells and tissues
AsIII species were previously quantified in cell lysates, but not tissue homogenates Stability of trivalent arsenicals in the cellular environment was not characterized
Hernandez-Zavala, A., et al., (2008) J. Anal. At. Spectrom. 23, 342-351.
13 of 43
Dissertation Hypothesis
Trivalent arsenicals can be quantified in biological systems and used as predictors of
the susceptibility to the diabetogenic effects of As exposure
14 of 43
Research Strategy
Laboratory-based study• Determination of AsIII retention and distribution in a mouse model
of As-induced diabetes
Population-based study• Characterization of AsIII retention
in a target human tissue and associations between this retention
and risk of diabetes
32
Optimization of HG-CT-AAS• Method optimization and validation
1
15 of 43
Overview
Introduction Hypothesis Results
Method optimization and validation Population-based study
Conclusions Questions
16 of 43
MAsIII and DMAsIII can be quantified and are stable in the reductive environment of cells and tissues.
HG-CT-AAS Validation
To characterize the presence and stability of methylated trivalent arsenicals, particularly toxic but unstable DMAsIII, in cell and tissue samples, using an optimized HG-CT-AAS system.
Hypothesis
17 of 43
Detection of AsIII species in mouse liver homogenate by HG-CT-AAS
0 10 20 30 40 50 600
0.1
0.2
0.3
0.4
0.5
0.6LF3df5LF3Cdf5
Time (s)
Abso
rban
ce
MAsIII+V
DMAsIII
MAsIIIiAsIII
10% liver homogenate from a mice exposed to 50 ppm As as iAsIII in water
iAsIII+V
DMAsIII+VDirect
Cys-treated
18 of 43
- + - + - + - +02468
1012
DIWHomogenate
iAs MAs DMAs
As,
ng
**
Cys
*
Validation of AsIII analysis in mouseliver homogenate
Direct analysis of unexposed mouse liver homogenates spiked with AsV and AsIII standards. Mean ± SD, n = 3, (*) Statistical difference between spiked DIW and homogenate p<0.05
AsV StandardsAsIII Standards
- + - + - + - +02468
1012
DIWHomogenate
iAs MAs DMAs
As,
ng
Cys
*
19 of 43
Recovery and stability of AsIII species inmouse liver homogenate
10% homogenate in DIW
Exposure to iAsIII in drinking water (50 ppm As) for 9 days
Aliquots for immediate analysis and storage at either -80°C or
0°C
Immediate Analysis Acid Digestion AnalysisStability after 1, 6, 13 and
22 days of storage at -80°C or 0°C.
Day: 0 1 6 13 22
Analysis of AsIII and AsIII+V species by HG-CT-AAS
20 of 43
Oxidation state specific quantification ofAs in mouse liver homogenate
Direct analysis of As species in mouse liver homogenate after exposure to 50 ppm As as iAsIII in drinking water for 9 days. n = 1 with 3 replicate measurements
iAs MAs DMAs Total As0
500
1000
1500
2000PentavalentTrivalent
ng
As/
g tis
sue ~ 65% of As measured is
trivalent:
iAsIII (8%)MAsIII (12%)
DMAsIII (45%)
21 of 43
Recovery of As species inmouse liver homogenate
iAs MAs DMAs Total0
20
40
60
80
100
120
Rec
over
y (%
)
Phosphoric acid digestion oxidizes AsIII species but does not change the methylation status.
Total recovery using direct hydride generation compared to phosphoric acid digestion is at least 95%.
High-affinity thiol binding of iAsIII could cause lower iAs recovery (~85%)
22 of 43
Stability of DMAsIII in mouse liver homogenate
0 2 4 6 8 10 12 14 16 18 20 22 240
200
400
600
800
1000
1200
1400
1600
1800DMAsIII
DMAsIII+V acid digestionDMAsIII+V
Storage at -80°C (days)
DM
As
(ng
As
/ g ti
ssue
)
0 2 4 6 8 10 12 14 16 18 20 22 240
200
400
600
800
1000
1200
1400
1600
1800DMAsIII
DMAsIII+V acid digestionDMAsIII+V
Storage at 0°C (days)
DM
As
(ng
As
/ g ti
ssue
)
* **
***
Direct analysis of As in mouse liver homogenate after exposure to 50 ppm As in drinking water for 9 days
Mean +/- SD, n = 3. (*) significant difference compared with immediate analysis, p<0.01
0°C-80°C
23 of 43
Generation of DMAsIII in cultured cells
Tri- and pentavalent As species were measured by HG-CT-AAS in cell
lysates before and after pretreatment with 2% cysteine (mean ± SD, n = 3).
UROtsa/F35 cell line expresses rat As3mt and was established because cultured urothelial cells do not methylate As
DMAsIII was generated in UROtsa/F35 cultures exposed to 0.1 mM MAsIII (15 ng As per well) for up to 18 hours
Cell Lysate
24 of 43
Stability of DMAsIII in UROtsa/F35 cell lysates
0 5 10 15 20 25
0.0
0.4
0.8
1.2
1.6
2.0
2.4DMAsIII -80°CDMAsV -80°C
* **
Storage at -80°C (days)
DM
As
in L
ysat
e (n
g)
0 5 10 15 20 25
0.0
0.4
0.8
1.2
1.6
2.0
2.4DMAsIII 0°CDMAsV 0°C
Storage at 0°C (days)
DM
As
in L
ysat
e (n
g)
(*) significant difference compared with immediate analysis, p<0.01 (Mean ± SD, n = 3. ) % recovery As: 90-100%
0°C-80°C
25 of 43
0.5 µM MAsIII exposure (18 hours) in UROtsa/F35 cell culture
Mock shipment conditions: Ice packs pre-frozen at -80°C; ( ) dry ice replenished at end of day two
(*) Significant decrease (p<0.05) compared with immediate analysis (one-way ANOVA), n = 3.
Stability of DMAsIII in UROtsa/F35 cell lysates under shipping conditions
0 1 2 3 4 5 6 7 80
1
2
3
4
5
6
7
8
Ice PacksDry Ice
**
*
*
*
Storage Time (Days)
DM
AsIII
(ng)
Cell Lysate
26 of 43
Summary
AsIII species can be quantified in mouse liver homogenates after exposure to iAsIII in drinking water.
In liver homogenates, DMAsIII is stable for at least 3 weeks when stored at -80°C, and DMAsIII is stable in
cell lysates prepared in water for at least 3 weeks when stored at 0°C or -80°C.
DMAsIII is stable for at least 2 days in cell lysates when stored in dry ice under mock shipping
conditions.
27 of 43
Conclusion
It is feasible to design studies systematically analyzing AsIII metabolites in laboratory- and population-based samples.
28 of 43
Overview
Introduction Hypothesis Results
Method optimization and validation Population-based study
Conclusions Questions
29 of 43
Analysis of AsIII Species in BECs
To examine the retention of tri- and pentavalent metabolites of iAs in urinary bladder exfoliated cells (BECs) isolated from residents of an As-endemic region of Mexico and determine the associations between diabetes and markers of As exposure in BECs and urine.
The level of AsIII species in BECs is higher in individuals who develop diabetes as a result of iAs exposure as compared to diabetes-free controls.
Hypothesis
30 of 43
Background
Hernández-Zavala, A., et al., (2008) Environ. Health Perspect. 116: 1656–1660.
Urine
BECs
As species detected in urine (a) and bladder exfoliated cells (BECs) (b) in residents of Zimapan, an arsenicosis-endemic area in Mexico
iAs in drinking water ranged from < 1 – 190 ppb
31 of 43
Study Design
31
Cross-Sectional Study
BECs are collected from spot urine samples of 378 individuals living in Chihuahua Shipped by 2-day carrier (on dry ice) to our lab every 4 weeks
BEC Isolation
Analyses iAs metabolites in BECs and urine iAs in drinking water Epigenetics: DNA methylation profiles in blood and BECs Metabolomics (urine and blood)
Residents in Chihuahua, Mexico are exposed to As in drinking water (0.1 - 400 ppb) Participants provide a spot urine undergo a medical exam to diagnose diabetes
Fasting plasma glucose (FPG) ≥ 126 mg/dL Two-hour plasma glucose (2HPG) ≥ 200 mg/dL after oral glucose tolerance test
(OGTT) Self-reported doctor’s diagnosis or use of anti-diabetic medication
32 of 43
Study Population Characteristics
a FPG or OGTT data are not available for four individuals and are excluded from the diabetes stratification analysis.
b Specific gravity was measured in 377 samples.(*) For continuous variables, a significant difference between diabetics and non-
diabetics by Student’s t-test, p < 0.05.0
Population Diabetica Non-diabeticMean (N) SD (%) Mean (N) SD (%) Mean (N) SD (%)
Population (378) (100) (66) (18) (308) (82)Female (255) (67) (44) (67) (211) (67)Age (years) 49.0 16.0 56* 12.0 48* 16.0 iAs in drinking water (ppb) 54.9 52.7 60.0 50.9 53.7 53.2 BMI 29.2 6.1 30.8* 5.4 28.9* 6.2 FPG (mg/dL) 95.9 39.5 155.7* 62.8 83.2* 12.1 2HPG (mg/dL) 118.6 60.4 204.9* 86.0 100.4* 31.1 Creatinine (mg/dL) 128.4 90.1 127.1 85.7 128.7 91.1Specific Gravityb 1.014 0.007 1.017* 0.008 1.014* 0.007
33 of 43
0 10 20 30 40 50 600
20,000
40,000
60,000
80,000
100,000DI Water
BEC Sample
Time (seconds)
Cou
nts
Per S
econ
dAnalysis of AsIII species in BECs
Quantify AsIII and AsV using HG-CT-ICP-MS
The HG-CT system is connected to an ICP-MS in the UNC-CH Biomarker Mass Spectrometry FacilityLimits of Detection: HG-CT-ICP-MS (0.04 – 2.04 pg); HG-CT-AAS (9- 26 pg)
34 of 43
As metabolites in BECs areassociated with urinary As species
xxx
-2 -1 0 1 2 3-4
-2
0
2
4
= .48, r2 = .26
Log iAs Urine (ng/mL)
Log
iAsIII
+V B
ECs
(pg
As/1
0,00
0 ce
lls)
-2 -1 0 1 2 3-4
-2
0
2
4
= .78, r2 = .41
Log MAs Urine (ng/mL)
Log
MAsIII
+V B
ECs
(pg
As/1
0,00
0 ce
lls)
-2 -1 0 1 2 3-4
-2
0
2
4
= .60, r2 = .14
Log DMAs Urine (ng/mL)
Log
DMAs
III+V
BEC
s(p
g As
/10,
000
cells
)
-2 -1 0 1 2 3-4
-2
0
2
4
= .67, r2 = .28
Log TAs Urine (ng/mL)
Log
TAsIII
+V B
ECs
(pg
As/1
0,00
0 ce
lls)
Sum of As Species
DMAsMAsiAs
35 of 43
Composition of As Species in BECsdiffers from that in urine
(***) Significant difference between BECs and urine based on one-way ANOVA, p < 0.05.
0
20
40
60
80
100BECsUrine
iAs MAs DMAs
*** ***%
of T
otal
As
36 of 43
Logistic Regression Analysis
Adjusted for age, gender, and BMI Units of odds ratio (OR) and 95% confidence interval (CI) are standardized to an
increment of one inter-quartile range (IQR) p-value for comparison of cases with non-diabetic individuals * Individuals self-reporting a doctor diagnosis or taking anti-diabetic medication but
not classified as diabetic by FPG or 2HPG are excluded.
Diabetes Classification
FPG ≥ 126 mg/dL2HPG ≥ 200 mg/dLn = 363*
Model 1 Model 2
Diabetes Classification
FPG ≥ 126 mg/dL2HPG ≥ 200 mg/dLSelf-report of doctor diagnosisuse of anti-diabetic medicationn = 374
37 of 43
iAsIII and MAsIII retained in BECs are associated with diabetes
DMAs/iAs
DMAs/MAs
MAs/iAs
III+VSum As
VDMAs
VMAs
ViAs
IIIDMAs
IIIMAs
IIIiAs **
*
*
OR with 95% CI
**
*
*
*
*
OR with 95% CI
Model 1 Model 2
BEC
s
Model 1: Diabetes = FPG≥126 mg/dL, 2HPG≥200 mg/dL, doctor’s diagnosis or use of anti-diabetic medication, n = 374. Model 2: Individuals self-reporting but not classified as diabetic by FPG or 2HPG are excluded, n = 363. (*) p < 0.05 for comparison of cases to non-diabetic individuals.
38 of 43
As metabolites in urine are only associated with diabetes after creatinine adjustment
iAs Drinking Water
III+VSum AsIII+VDMAsIII+VMAsIII+ViAs
Specific Gravity
III+VSum AsIII+VDMAsIII+VMAsIII+ViAs
Creatinine
DMAs/iAsDMAs/MAs
MAs/iAsIII+VSum AsIII+VDMAsIII+VMAsIII+ViAs
*
OR with 95% CI
*
****
*
OR with 95% CI
Model 1 Model 2
Urin
eA
djus
ted
Urin
e
39 of 43
Summary
As metabolites retained in BECs are associated with the concentration of As species in urine.
Trivalent, but not pentavalent As species in BECs are positively associated with diabetes risk, while the DMAs/MAs and DMAs/iAs ratios are negatively associated with diabetes risk.
iAs is primarily retained in BECs, while DMAs is primarily excreted in urine.
40 of 43
Conclusions
Compositional differences in the As metabolite profiles in urine and BECs indicate that the urinary profiles of As metabolites do not reflect As speciation in a target tissue.
Trivalent As species in BECs can be used as markers of diabetes risk in individuals exposed to iAs, thus avoiding bias associated with measures of As species in urine.
41 of 43
Overview
Introduction Hypothesis Results
Method optimization and validation Population-based study
Conclusions Questions
42 of 43
The use of trivalent arsenical quantification in biological systems as sensitive biomarkers for the risk assessment of iAs-
associated diseases, including diabetes mellitus.
Contributions to the field of toxicology
A robust HG-CT-based technique for the quantification of trivalent
arsenicals in a variety of biological samples.
43 of 43
Acknowledgements
GIL grant 200710.0028NIH grants DK056350 and 5R01ES015326Curriculum in Toxicology Predoctoral Traineeship National Research Service Award T32 ES0071260
Miroslav Styblo, PhDRebecca Fry, PhDJames Samet, PhD, MPHJames Swenberg, DVM, PhDDavid Thomas, PhD
UNC Chapel Hill
Curriculum in ToxicologyBBSP 2008 CohortMarila Cordeiro-Stone, PhDDepartment of NutritionLan Ding, PhDChristelle Douillet, PhDZuzana Drobna, PhDMichelle Mendez, PhDDavid Paul, PhDJesse Saunders, BAFelecia Walton, BSBiomarker Mass Spectrometry FacilityWanda Bodnar, PhDPeter Cable, BSLeonard Collins, BS, MBA
Jiří Dědina, PhDTomáš Matoušek, PhDMilan Svoboda, PhD
Committee Members
Institute of Analytical Chemistry ofthe ASCR Funding
Dana Loomis, PhDTeam in Mexico led by Carmen González Horta and Gonzalo García-Vargas
Additional Collaborators
Mary Z. Kyprianou, PhD, BCIA Fellow
POTS Treatment Center
44 of 43
First Author Publications
Currier, J.M., Saunders, R.J., Ding, L., Bodnar, W.M., Cable, P., Matousek, T., Creed, J., and Styblo, M. (2013) “Comparative oxidation state specific analysis of arsenic species by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry and hydride generation-cryotrapping-atomic absorption spectrometry.” Under review, Journal of Analytical Atomic Spectrometry 28, 843-852.
Currier, J.M., Svoboda, M., Matousek, T., Dedina, J., and Styblo, M. (2011) “Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues.” Metallomics 3, 1347-1354.
Currier, J.M., Svoboda, M., de Moraes, D.P., Matousek, T., Dedina, J., and Styblo, M. (2011) “Direct analysis of methylated trivalent arsenicals in mouse liver by hydride generation-cryotrapping-atomic absorption spectroscopy.” Chemical Research in Toxicology 24, 478-480.
45 of 43
Co-Authored Publications Related toDissertation Research
Matousek, T., Currier, J.M., Trojankova, N., Saunders, R.J., Ishida, M.C., González-Horta, C., Musil, S., Mester, Z., Styblo, M., and Dedina, J. (2013) “Selective hydride generation- cryotrapping- ICP-MS for arsenic speciation analysis at picogram levels: analysis of river and sea water reference materials and human bladder epithelial cells.” Submitted, Journal of Analytical Atomic Spectrometry.
Douillet, C., Currier, J.M., Saunders, R.J., Bodnar, W.M., Matousek, T., and Styblo, M. (2013) “Methylated trivalent arsenicals are potent inhibitors of glucose stimulated insulin secretion by murine pancreatic islets.” Toxicology and Applied Pharmacology 267, 11-15.
Del Razo, L.M., Garcia-Vargas, G.G., Valenzuela, O.L., Hernandez Castellanos, E., Sanchez-Pena, L.C., Currier, J.M., Drobna, Z., Loomis, D., and Styblo, M. (2010) “Exposure to arsenic in drinking water and prevalence of diabetes in the Zimapan and Lagunera regions in Mexico.” Environmental Health 10:73.
46 of 43
Additional Co-Authorships andManuscripts in Preparation
Currier, J.M., González-Horta, C., Del Razo, L.M., Sánchez-Ramírez, B., Ballinas-Casarrubias, L., García-Vargas, G., Ishida, M.C., Saunders, R.J., Drobna, Z., Loomis, D., and Styblo, M. (2013) “Trivalent Arsenicals in Exfoliated Bladder Cells - Novel Biomarkers of Diabetes Risk Associated with Chronic Exposure to Inorganic Arsenic.” Submitted to Environmental Health Perspectives.
Currier, J.M., Saunders, R.J., Drobna, Z., Douillet, C., and Styblo, M. (2013) “Oxidation state specific analysis of arsenic species in tissues of wild-type and arsenic (+3 oxidation state) methyltransferase (As3mt) knockout mice.” In Preparation.
Cheng, W-Y., Currier, J.M., Bromberg, P.A., Silbajoris, R., Simmons, S.O., and Samet, J.M. (2012) “Linking oxidative events to inflammatory and adaptive gene expression induced by exposure to an organic PM component.” Environmental Health Perspectives 120, 267-274.
Ariyananda, L., Antonopoulos, C., Currier, J., and Colman, R.F. (2011) “In vitro hybridization and separation of hybrids of human adenylosuccinate lyase for wild type and disease-associated mutant enzymes.” Biochemistry 50, 1336-1346.
47 of 43
Future Directions
•Optimization of HPLC-based separation techniques for quantification of trivalent and thiolated arsenicals. 1
•Characterization of the role that methylation plays on the development of As-induced diabetes using WT and As3mt-KO mice.
2
•Prospective epidemiology studies looking at new diabetes cases over time to strengthen the evidence for an association between iAs exposure and development of diabetes.
3
48 of 43
Specific Aim 1: Extra Slides
1
49 of 43
Waste
Reaction Coil
NaBH4
Air
Gas-liquidseparator
Cryotrap
He, H2
As LampHeating System
AASDetector
Multi-Atomizer
Liquid N2
U-tube3-wayvalve
HG-CT-AAS
Tris-Buffer
Sample +H2O
FIAS Remote Activation
Detection Limits: 9-20 pg
193.7 nm
Peristaltic Pump
Detection Limits (HG-CT-ICP-MS):
0.04-2.04 pg
50 of 43
Detection Limit iAsIII MAsIII DMAsIII iAsIII+V MAsIII+V DMAsIII+V
Instrumental (pg As) (a) 14 13 9 10 10 12Tissue (ng As/g tissue) (b) 6 5 4 4 4 5
(a) The instrumental detection limits were calculated from the AAS spectra generated for blanks (control liver homogenates, n=8) as 3(SD/slope) for the absorbance areas with the retention times corresponding to arsine, methylarsine, and dimethylarsine signals.
(b) Tissue detection limits were calculated from the instrumental detection limits and reflect the concentration and dilution of the liver homogenates used for the analysis.
Instrumental and tissue detection limits for analysis of AsIII and AsIII+V species in mouse liver homogenate
51 of 43
Matrices As Standard Linear Regression (b) Correlation CoefficientDIW iAsV 0.821x - 0.019 0.999
MAsV 0.825x - 0.024 0.999DMAsV 0.835x - 0.024 0.999
Homogenate iAsV 0.873x + 0.00004 0.999MAsV 0.880x - 0.005 0.998
DMAsV 0.941x - 0.027 0.997
(a) Arsines and methyl substituted arsines were generated after 1-hour pretreatment with 2% L-cysteine.(b) The linear regression for each AsV standard was determined over the range of 0.125 to 4 ng As/mL.
Characteristics of the calibration curves for AsV standards spiked into DIW or 10% liver homogenate
52 of 43
As speciesAnalysis of digested
liver homogenateng As/g of tissue
Direct analysis of freshliver homogenate
ng As/g of tissue % Recovery
iAsIII 151 ± 15iAsV 158 ±16iAsIII+V 392 ± 12 309 ± 7 79 ± 3MAsIII 220 ± 5MAsV 143 ± 6MAsIII+V 390 ± 1 363 ± 4 93 ± 1DMAsIII 828 ± 11DMAsV 324 ± 16DMAsIII+V 1069 ± 10 1152 ± 11 108 ± 2
Total AsIII+V 1851 ± 16 1824 ± 14 99 ± 1
The concentration of As species in mouse liver homogenate
53 of 43
Stability of AsIII species in mouse
liver homogenate
The direct HG-CT-AAS analysis was used to determine the concentrations of iAs (A, B), MAs (C, D) and DMAs (E, F) species in aliquots of the fresh homogenate and in aliquots stored at –80°C (A, C, E) or 0°C (B, D, F) for up to 22 days (mean ± SD, n = 3). To control for As recoveries during the direct analyses, iAsIII+V, MAsIII+V, and DMAsIII+V were determined in aliquots of the fresh homogenate digested in phosphoric acid (mean, n = 3). *The concentration is significantly different from that found in the fresh homogenate (p < 0.01).
0 2 4 6 8 10 12 14 16 18 20 22 240
200400600800
10001200140016001800
DMAs I I I
DMAsV
DMAs I I I +V acid digestionDMAs I I I +V
* *
Storage at -80°C (days)
DMAs
(ng
As /
g ti
ssue
)
0 2 4 6 8 10 12 14 16 18 20 22 240
200400600800
10001200140016001800
DMAs I I I
DMAsV
DMAs I I I +V acid digestionDMAs I I I +V
*
**
*
Storage at 0°C (days)
DMAs
(ng
As /
g ti
ssue
)
0 2 4 6 8 10 12 14 16 18 20 22 240
100
200
300
400
500
600MAs I I I
MAsV
MAs I I I +V acid digestionMAs I I I +V
* * *
Storage at -80°C (days)
MAs
(ng
As
/ g
tissu
e)0 2 4 6 8 10 12 14 16 18 20 22 24
0
100
200
300
400
500
600MAs I I I
MAsV
MAs I I I +V acid digestionMAs I I I +V
**
Storage at 0°C (days)
MAs
(ng
As
/ g
tissu
e)0 2 4 6 8 10 12 14 16 18 20 22 24
0
100
200
300
400
500
600iAs I I I
iAsV
iAs I I I +V acid digestioniAs I I I +V
* *
Storage at -80°C (days)
iAs
(ng
As /
g ti
ssue
)
0 2 4 6 8 10 12 14 16 18 20 22 240
100
200
300
400
500
600iAs I I I
iAsV
iAs I I I +V acid digestioniAs I I I +V
Storage at 0°C (days)iA
s (n
g As
/ g
tiss
ue)
A
C
E
B
D
F
54 of 43
Generation of DMAsIII in UROtsa/F35 cell
cultures
Generation of DMAsIII in UROtsa/F35 culture exposed to 0.1 mM MAsIII (15 ng As per well) for up to 18 hours. Tri- and pentavalent As species were measured by HG-CT-AAS in culture medium (A) and cell lysates (B) before and after pretreatment with 2% cysteine (mean ± SD, n = 3).
Lysates
Media
55 of 43
TritonX100 oxidizes DMAsIII
Effect of Triton X100 on DMAsIII stability in cell lysates: UROtsa/F35 cells were exposed to 0.1 μM iAsIII (15 ng As/well) for 24 hours. Cells were then lysed in either DIW or 0.5% Triton X-100. DMAsIII and DMAsV were analyzed in fresh cell lysates by HG-CT-AAS. Values represent the percentage of total As in lysate (mean ± SD, n=3). * The percentage of DMAsIII in lysates prepared in Triton X100 is significantly different from that in lysates prepared in DIW (p < 0.01).
56 of 43
Specific Aim 1b: Extra Slides
1b
57 of 43
Effectiveness of HPLC and HG-CT
Column:Mobile Phase: Flow Rate: Injection Vol:
HPLC-ICP-MS1 Agilent 1260 Infinity HPLC with 7500cx ICP-MS
Masses:Integration:RF Power:Plasma Gas:Carrier Gas:Make-up Gas:Cell Gas: Nebulizer:
0 50 100 150 200 250 300 3500
2.0×10 4
4.0×10 4
6.0×10 4
8.0×10 4
iAs I I I
MAs I I I
DMAsVMAsV
DMTAV
DMAs I I I
iAsV
Time (s)
Inte
nsity
75As
(cp
s)75 (As), 77 (ArCl)0.1 s1550 WAr; 15 L/min Ar; 0.95 L/minAr; 0.25 L/minHe; 4.0 L/minMicromist
Phenomenex Prodigy 3μ ODS(3) 100A, 150x4.60 mm (30°C)4.7 mM tetrabutylammonium hydroxide, 2 mM malonic acid,and 4% methanol (pH 5.85)1.5 mL/min20 µL
1) S. Rabieh, A. V. Hirner and J. Matschullat. J. Anal. At. Spectrom., 2008, 23, 544-549.
58 of 43
Effectiveness of HPLC and HG-CT
HG-CT-AAS1 Perkin-Elmer FIAS 400 with AAnalyst 800
Sample Vol: 500 µLBuffer: 0.75 M TRIS-HCl (pH 6)Reducing Agent: 1% NaBH4 in 0.1% KOHColumn Packing: Chromosorb WAW-DCMSColumn Heating: Ni80/Cr20 wire, 15 Ω
Carrier Gases: He (75 mL/min); H2 (15 mL/min)
Lamp: As electrodeless discharge (390 mA, 197.3 nm)
Atomizer: Multiatomizer (900°C)
1) T. Matousek, et al. Spectrochim. Acta Part B At. Spectrosc., 2008, 63, 396-406.
59 of 43
In vitro methylation:iAsIII as substrate for AS3MT
Representative HG-CT-AAS (A) and HPLC-ICP-MS (B) chromatograms of the in vitro methylation mixtures incubated with 1 μM iAsIII (i.e., 11.25 ng As). The mixture was analyzed directly or after reduction with cysteine or oxidation with 3% H2O2 for 2 hours
0 50 100 150 200 250 300 3500
5.0×10 4
1.0×10 5
1.5×10 5
DirectOxidized
Time (s)
Inte
nsity
75As
(cps
)
0 10 20 30 40 50
0.0
0.2
0.4
0.6AsI I I
AsI I I +V
Time (s)
Abso
rban
ce
A B
iAsI I I
DMAsV
DMAs I I IiAsViAs
DMAs
60 of 43
In vitro methylation:iAsIII as substrate for AS3MT
0
2
4
6
8
10AsI I I
AsV
AsI I I+V
iAs MAs DMAs
ng A
s/re
actio
n
02468
101214
25%
95%72%
TAsIII+V
ng A
s/re
actio
nA B
The in vitro methylation mixtures incubated with 1 μM iAsIII (i.e., 11.25 ng As) were analyzed directly or after reduction with cysteine or oxidation with 3% H2O2 for 2 hours: (A) the amounts of AsIII, AsV, and sums of AsIII + AsV species detected by HPLC-ICP-MS and HG-CT-AAS in the in vitro methylation mixture and (B) %As recovered by each technique (mean ± SD, n = 4).
61 of 43
In vitro methylation:MAsIII as substrate for AS3MT
0 50 100 150 200 250 300 3500
5.0×104
1.0×105
1.5×105
2.0×105
DirectOxidized
Time (s)
Inte
nsity
75As
(cps
)0 10 20 30 40 50
0.0
0.2
0.4
0.6
0.8AsI I I
AsI I I +V
Time (s)
Abso
rban
ce
A B DMAsV
MAsvMAs
DMAs
iAs iAsVDMAsIII
MAsIII
iAsIII
Representative HG-CT-AAS (A) and HPLC-ICP-MS (B) chromatograms of the in vitro methylation mixtures incubated with 1 μM MAsIII (i.e., 11.25 ng As). The mixture was analyzed directly or after reduction with cysteine or oxidation with 3% H2O2 for 2 hours
62 of 43
In vitro methylation:MAsIII as substrate for AS3MT
0
2
4
6
8
10
12AsI I I
AsV
AsI I I+V
iAs MAs DMAs
ng A
s/re
actio
n
02468
10121416
19%
83%99%
Sum AsIII+V
ng A
s/re
actio
nA B
The in vitro methylation mixtures incubated with 1 μM MAsIII (i.e., 11.25 ng As) were analyzed directly or after reduction with cysteine or oxidation with 3% H2O2 for 2 hours: (A) the amounts of AsIII, AsV, and sums of AsIII + AsV species detected by HPLC-ICP-MS and HG-CT-AAS in the in vitro methylation mixture and (B) %As recovered by each technique (mean ± SD, n = 4).
63 of 43
AsIII species interact with AS3MT
0 100 200 300 4000
200000
400000
600000
800000
Inte
nsity
75As
(cps
)
0 100 200 300 4000
200000
400000
600000
800000
Inte
nsity
75As
(cps
)
0 100 200 300 4000
30000
60000
90000
120000
Time (s)
Inte
nsity
75As
(cps
)
60 70 800
200000
400000
600000
Time (s)
Inte
nsity
75As
(cps
)
80 90 1000
200000
400000
600000
800000
Time (s)
Inte
nsity
75As
(cps
)
0 100 200 300 4000
20000400006000080000
100000120000
Inte
nsity
75As
(cps
)
HPLC-ICP-MS profiles for the assay mixture (A; SAM, TCEP, TRIS-HCL), 0.5 μM iAsIII (A), MAsIII (B), or DMAsIII (C) standards spiked into the methylation mixture in the absence (black line) or presence (blue line) of AS3MT (60 µg/mL) or spiked into the TRIS-HCl buffer alone (green line).
A B
C D
64 of 43
AsIII species interact with AS3MT
Ultrafiltration of 1 μM iAsIII (A), MAsIII (B), or DMAsIII (C) in the absence (-) or presence (+) of AS3MT (60 µg/mL, mean ± SD, n = 3). Values with the same symbol indicate a significant difference due to the effect of AS3MT (a,b) or filtering (#) on the recovery of As (p < 0.05).
0
5
10
15
20
-FilteredAS3MT - -+ +
+ +-
iAs,
ng/
reac
tion
0
5
10
15
20
a
b,#a,#
b
-FilteredAS3MT - -+ +
+ +-
MAs,
ng/
reac
tion
0
5
10
15
20a
b,#
a,#b
-FilteredAS3MT - -+ +
+ +-
DMAs
, ng/
reac
tion
A B C
DMAsIIIMAsIIIiAsIII
65 of 43
Urine impairs the detection ofDMAsIIIby HPLC-ICP-MS
0 100 200 300 4000
250005000075000
100000125000150000
DMAsIII in TRIS
Unspiked urineDMAsIII in urine
Time (s)
Inte
nsity
75As
(cps
)
The HPLC-ICP-MS profiles for 0.5 µM DMAsIII standard spiked into 100 mM TRIS-HCl buffer (pH 7.4) or human urine from an unexposed subject.
Peak Area
Reduction73%
66 of 43
Speciation detection methods Separation of arsenic metabolites:
HPLC or LC Hydride Generation – Cryotrapping (HG-CT)
Coupled with: Atomic emission spectroscopy (AES) Atomic fluorescence spectrometry (AFS) Atomic absorption spectrometry (AAS) Inductively coupled plasma-mass spectrometry
(ICP-MS) Electronspray ionization-MS (ESI-MS, ESI-MS-MS)
67 of 43
Specific Aim 2: Extra Slides
2
68 of 43
Specific Aim 2
To compare the internal dose of trivalent arsenicals in target organs of WT and As3mt knockout mice after iAsIII exposure, and to determine exposures that produce equivalent internal doses in tissues regulating glucose homeostasis (skeletal muscle, adipose tissue, pancreas, and liver).
iAsIII is highly retained in KO mouse tissues, and MAsIII and DMAsIII are extensively retained in tissues from
WT mice regulating glucose homeostasis.
Hypothesis
69 of 43
Diabetes
Arsenic
AS3MT ?
1. Drobna, Z., et al., (2009) Chem. Res. Toxicol., 22, 1713–1720.2. Hughes, M.F., et al., (2010) Toxicol Appl Pharmacol., 249, 217-
23.3. Chen, B., et al., (2011) Toxicological Sciences., 124, 320–326.
o Arsenic (3+ Oxidation State) Methyltransferase (As3mt) Knockout Miceo Can be used to compare the effects of
methylation on the development of diabetes using WT mice exposed to 50 ppm As
o This genotype affects the retention and methylation of As.1-3
o Arsenicals retained in RBCs, liver, kidney and lung of As3mt-KO mice were higher than in WT mice after exposure to iAsIII; however, As in plasma was higher in WT mice.3
o Oxidation state specific speciation of As has not yet been studied in these tissues
Specific Aim 2: Background
70 of 43
15 ppm As 20 ppm As
As3mt-KO Mice
25 ppm As
Male As3mt-KO and C57BL/6 mice(As3mt-KO: n = 5/treatment; WT: n = 10)
Quantify AsIII and AsV by direct analysis of tissue homogenates by HG-CT-AAS
4 Weeks
Animals are maintained on a grain-based diet and exposed to As as arsenite (iAsIII) in drinking
water
Liver, Pancreas, Skeletal Muscle, Adipose Plasma, RBCs, Testes, Brain, Heart, Kidney, Lung, Intestine
WT Mice*
30 ppm As
0 ppm As 50 ppm As
Examine As recoveries indigested tissues
Specific Aim 2: Experimental design
* As-induced diabetes
mouse model
71 of 43
Plasma
Plasma As from WT mice exposed to 50 ppm As is >10-fold higher than KO mice exposed to 0 – 30 ppm As
0 15 20 25 30 500
255075
100125325350375
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
* * * * *
iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVO
As, n
g/m
L
72 of 43
Sum of As in liver of WT mice (50 ppm) correspondsto between15-25 ppm in As3mt-KO mice
Liver
0 15 20 25 30 500
2,000
4,000
6,000
8,000
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
*
* iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVOAs, n
g/g
tissu
e
73 of 43
Sum of As in WT mice (50 ppm) is equivalent to a30 ppm As exposure in As3mt-KO mice
Pancreas
0 15 20 25 30 500
200
400
600
800
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
*
* **
iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVOAs, n
g/g
tissu
e
74 of 43
Sum of As in WT mice (50 ppm) corresponds to a25-30 ppm exposure in As3mt-KO mice
Skeletal Muscle
0 15 20 25 30 500
200
400
600
800
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
*
* *
iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVOAs, n
g/g
tissu
e
75 of 43
Sum of As in WT (50 ppm) mice corresponds to a15-30 ppm exposure in As3mt-KO mice.
Adipose Tissue
0 15 20 25 30 500
100
200
300
400
500
600
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
*
iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVOAs, n
g/g
tissu
e
76 of 43
% Recovery of As by direct analysis
a For As3mt-KO treatment groups, %recovery is for iAsIII+V
b For WT mice treated with 50 ppm As, %recovery is for total speciated As
c Mean±SD; As3mt-KO, n = 5 and WT, n = 10
As3mt-KOa WTb
Tissuec 15 ppm 20 ppm 25 ppm 30 ppm 50 ppm Pancreas 106±19 89±1 90±7 92±7 103±11
Liver 119±18 112±27 106±13 108±17 119±38Muscle 52±10 66±20 60±6 63±12 85±15Adipose 92±50 100±45 115±96 104±14 109±47
77 of 43
Specific Aim 2: Conclusions
Trivalent arsenic species can be quantified by HG-CT-AAS in tissues critical for glucose homeostasis from mice exposed to iAsIII. High recoveries are achieved for liver, adipose, and pancreas.
For tissues critical to glucose homeostasis, doses of 25 and 30 ppm As as iAsIII in As3mt-KO mice will produce equivalent total As retention to that of WT mice.
MAsIII and DMAsIII were extensively retained in tissues of WT mice, while iAsIII and iAsV were predominantly retained in tissues of As3mt-KO mice.
78 of 43
Water consumption and body weights
0 1 2 3 422
24
26
28
30
32
34
360 ppm As3mt-KO15 ppm As3mt-KO20 ppm As3mt-KO25 ppm As3mt-KO30 ppm As3mt-KO50 ppm WT
Time (weeks)
Body
Wei
ght (
g/an
imal
)
1 2 3 40
1
2
3
4
5
Time (Weeks)
H 2O
Cons
umpt
ion
(mL/
anim
al/d
ay)
15 20 25 30 500
25
50
75
100
125
150
As3mt-KO WTppm ppm ppm ppm ppm
iAs
inta
ke (µ
g/an
imal
/day
)
A B
C Average daily water consumption (A) and body weights (B) for As3mt-KO mice exposed to 0 (●), 15 (■), 20 (▲), 25 (♦), or 30 (▼) ppm As and WT mice exposed to 50 ppm As (○). Daily iAs intake (C) was estimated from average daily water consumption (mean+SD, As3mt-KO, n = 5;WT, n = 10).
79 of 43
Intestine with content
0 15 20 25 30 500
4,000
8,000
12,00020,00024,00028,000
As3mt-KO WT
ppm ppm ppm ppm ppm ppm*
**
*
*
iAsIII
iAsV
MAsIII
MAsV
DMAsIII
DMAsV
TMAsVOAs, n
g/g
tissu
e
Sum of As in WT (50 ppm) mice corresponds to 15-30 ppm exposure in As3mt-KO mice.
80 of 43
WT and As3mt-KO mice
0 15 20 25 30 500
200
400
600
800
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
Blood Cells
*
* * **
As, n
g/g
tissu
e
0 15 20 25 30 500
200
400
600
800
1,000
1,200
1,400
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
Brain
*
* *
* *
As, n
g/g
tissu
e
0 15 20 25 30 500
200
400
600
800
1,000
1,200
1,400
1,600
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
*
*
Testes
As, n
g/g
tissu
e
iAsIII
iAsVMAsIII
MAsVDMAsIII
DMAsV TMAsVOLegend:
81 of 43
WT and As3mt-KO mice
0 15 20 25 30 500
1,000
2,000
3,000
4,000
5,000
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
Kidney
*
As, n
g/g
tissu
e
0 15 20 25 30 500
500
1,000
1,500
2,000
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
Lung
*
* *
* *
As, n
g/g
tissu
e
0 15 20 25 30 500
200
400
600
800
1,000
1,200
1,400
As3mt-KO WT
ppm ppm ppm ppm ppm ppm
Heart
*
* *
As, n
g/g
tissu
e
iAsIII
iAsVMAsIII
MAsVDMAsIII
DMAsV TMAsVOLegend:
82 of 43
% Recovery of As by direct analysis
a For As3mt-KO treatment groups, %recovery is for iAsIII+V
b For WT mice treated with 50 ppm As, %recovery is for total speciated Asc Mean±SD; As3mt-KO, n = 5 and WT, n = 10
As3mt-KOa WTb
Tissuec 15 ppm 20 ppm 25 ppm 30 ppm 50 ppm Pancreas 106±19 89±1 90±7 92±7 103±11
Liver 119±18 112±27 106±13 108±17 119±38Muscle 52±10 66±20 60±6 63±12 85±15Adipose 92±50 100±45 115±96 104±14 109±47
Blood Cells 133±10 139±19 142±18 148±22 128±23Intestine 106±21 162±31 179±121 125±27 114±49Kidney 123±23 81±4 112±59 98±50 63±17Lung 95±14 121±32 128±24 131±31 102±45Heart 71±8 78±6 77±7 89±7 124±23Brain 83±11 80±8 83±10 101±14 120±16Testes 114±49 178±43 123±55 66±9 156±66
83 of 43
Comparison of internal doses
a Total speciated As is the sum of iAsIII+V, MAsIII+V, DMAsIII+V and TMAsVO determined by HG-CT-AAS (mean±SD; As3mt-KO, n = 5 and WT, n = 10) # Statistically non-significant difference in total speciated As compared to 50 ppm WT group (p > 0.05)
As3mt-KO WT
0 ppm 15 ppm 20 ppm 25 ppm 30 ppm 50 ppm
Pancreas 16±7 381±38 394±86 452±26 576±83# 693±141
Liver 67±10 3137±476# 3525±787# 4497±300# 5564±1024 3511±1174
Muscle 69±102 386±55 479±49 677±114# 688±147# 658±185
Adipose 21±14 181±107# 304±112# 413±329# 277±61# 239±118
Total speciated Asa (ng As/g tissue)
84 of 43
Specific Aim 3: Extra Slides
3
85 of 43
Metabolite RangesBECs (pg As/10,000 cells) Min. 25th Median 75th Max. Mean SDiAsIII 0.04 2.05 8.12 17.65 1807 23.82 102MAsIII 0.01 0.44 1.76 4.02 151.7 4.14 11.16DMAsIII 0.01 0.16 0.40 1.47 141.3 2.7 9.53iAsV 0.001 1.27 4.52 22.46 728.7 34.45 85.86MAsV 0.0003 0.19 0.85 4.87 776.2 12.98 52.21DMAsV 0.001 0.66 1.86 13.67 2303 48.67 199.7Sum AsIII+V 0.78 9.94 24.91 74.5 3773 126.4 357.9MAs/iAs ratio 0.01 0.15 0.2 0.28 3.64 0.26 0.28DMAs/MAs ratio 0.04 0.55 1.10 2.87 51.47 2.14 3.63DMAs/iAs ratio 0.004 0.10 0.20 0.50 35.0 0.77 2.5MAs+DMAs/iAs ratio 0.02 0.29 0.40 0.78 35.63 1.03 2.66Urine (ng As/mL) iAsIII+V 0.02 0.92 4.61 10.21 119.2 7.4 10.4MAsIII+V 0.03 2.21 7.31 15.97 131.1 11.1 13.0DMAsIII+V 0.36 12.24 40.38 82.73 307.2 55.12 53.8Sum AsIII+V 0.52 15.62 53.23 108.4 492.5 73.61 73.27MAs/iAs ratio 0.10 1.28 1.64 2.12 199.4 4.90 19.1DMAs/MAs ratio 1.73 4.09 5.18 7.05 86.2 6.22 5.25DMAs/iAs ratio 0.41 6.43 9.25 12.45 2117 31.0 144.4MAs+DMAs/iAs ratio 0.51 7.96 10.94 14.59 2317 35.94 162.6Drinking Water# 0.01 5.88 48.41 83.72 275.4 54.9 52.7
# iAs in drinking water (ng iAs/mL) was measured in 300 samples.
86 of 43
biAsII
I
biAsV
bMAsIII
bMAsV
bDMAsIII
bDMAsV
bTAsIIIV
0
500
1000
1500
2000
2500
3000
3500
TrivalentPentavalent
Total AsIII+V
pg A
s/10
,000
cel
ls
As species retained in BECs
As Species
Range(pg As/10,000
cells)
#<LOD
iAsIII 0.0405 – 1806.7 24iAsV 0.0011 – 728.7 11
MAsIII 0.0092 – 151.7 8MAsV 0.0003 – 151.7 7
DMAsIII 0.0107 – 141.3 2DMAsV 0.0011 – 2128.7 3Sum 0.7835 – 3136.9 0
For statistical purposes, samples below the limit of detection are replaced with LOD/√2
iAs Mas DMAs TAs
87 of 43
biAsIII
biAsV
bMAsIII
bMAsV
bDMAsIII
bDMAsV
bTAsIIIV
0
100
200
300
400
500
TrivalentPentavalent
Total AsIII+V
pg A
s/10
,000
cel
ls
As species retained in BECs
As Species
Range(pg As/10,000
cells)
#<LOD
iAsIII 0.0405 – 1806.7 24iAsV 0.0011 – 728.7 11
MAsIII 0.0092 – 151.7 8MAsV 0.0003 – 151.7 7
DMAsIII 0.0107 – 141.3 2DMAsV 0.0011 – 2128.7 3Sum 0.7835 – 3136.9 0 iAs Mas DMAs
TAsMean +/- SD, for statistical purposes, samples below
the limit of detection are replaced with LOD/√2
88 of 43
Association between total speciated As in BECs and urine
-2 -1 0 1 2 3-4
-2
0
2
4
= .88, r2 = .55
Log Sum As Urine (ng/mL)
Log
Sum
AsIII
BEC
(pg
As/1
0,00
0 ce
lls)
-2 -1 0 1 2 3-4
-2
0
2
4
= .52, r2 = .12
Log Sum As Urine (ng/mL)
Log
Sum
AsV B
EC(p
g As
/10,
000
cells
)
AsIII
AsV
Sum As Species
89 of 43
As species in BECs are associated
with iAs in drinking water
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .43, r2 = .53
Log iAs Water (ng/mL)
Log
iAs
III B
EC(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .21, r2 = .08
Log iAs Water (ng/mL)
Log
iAs
V BE
C(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .39, r2 = .45
Log iAs Water (ng/mL)
Log
MAs
III B
EC(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .27, r2 = .10
Log iAs Water (ng/mL)
Log
MAs
V BE
C(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .28, r2 = .21
Log iAs Water (ng/mL)Lo
g DM
AsIII
BEC
(pg
As/1
0,00
0 ce
lls)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .21, r2 = .07
Log iAs Water (ng/mL)
Log
DMAs
V BE
C(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .42, r2 = .54
Log iAs Water (ng/mL)
Log
Sum
As
III B
EC(p
g As
/10,
000
cells
)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .22, r2 = .09
Log iAs Water (ng/mL)
Log
Sum
As
V BE
C(p
g As
/10,
000
cells
)
A B
C D
E F
G H
The associations between logarithmically transformed concentrations of iAs in water and iAs (A,B), MAs (C,D), DMAs (E,F) and sum of As species (G,H) in BECs. Results of linear regression analysis are presented as slopes (ß) and coefficient of determination (r2). All slopes are significantly non-zero, p < 0.05.
90 of 43
iAs metabolites in urine are associated with iAs in drinking water
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .40, r2 = .45
Log iAs Water (ng/mL)
Log
iAs
Urin
e(n
g/m
L)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .41, r2 = .64
Log iAs Water (ng/mL)
Log
MAs
Urin
e(n
g/m
L)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .39, r2 = .67
Log iAs Water (ng/mL)
Log
DMAs
Urin
e(n
g/m
L)
-3 -2 -1 0 1 2 3-4
-2
0
2
4
= .40, r2 = .68
Log iAs Water (ng/mL)
Log
Sum
As
Urin
e(n
g/m
L)
A
B
C
D
Results of linear regression analysis for iAs (A), MAs (B), DMAs (C), and sum of speciated As (D) in urine compared with iAs in drinking water are presented as slopes (ß) and coefficient of determination (r2). All slopes are significantly non-zero, p < 0.05.
91 of 43
Females excrete more cells, but retain less As
0
100,000
200,000
300,000
400,000
500,000
600,000
Male Female
BEC
Coun
t
A
0
100
200
300
400
Male FemaleSu
m A
sIII+V
BEC
s(p
g/10
,000
cel
ls)
B
Gender differences in cells counts (A) and As retention (B) in BECs. Values are presented as mean ± SEM, n = 378.
92 of 43
Possible dilution effect in samples from males
2 3 4 5 6 7 8-1
0
1
2
3
4
Log BEC Count
Log
Sum
As
III+V
BE
Cs
(pg/
10,0
00 c
ells
)
2 3 4 5 6 7 8-1
0
1
2
3
4
Log BEC Count
Log
Sum
As
III+V
BE
Cs
(pg/
10,0
00 c
ells
)
A
B
ß = -.70, r2 = 0.54
ß = -.35, r2 = 0.12
The associations of cell counts with sum of As species retained in BECs for samples obtained from males (A) and females (B). Results from linear regression analysis are presented as slopes (ß) and coefficient of determination (r2). All slopes are significantly non-zero, p < 0.05.
Female
Male
93 of 43
Gender differences
0
20
40
60
80
100
iAsI I I
**
**
**
**
**
iAsV MAsII I MAsV DMAI I I DMAV
% o
f Tot
al A
s
0
20
40
60
80
100MaleFemale
iAs MAs DMAs
**
**
% o
f Tot
al A
s
Gender differences in percent composition of As metabolites retained in BECs and excreted in urine. Values for each arsenical represent percent of the sum of speciated As (mean ± SD, n = 378). (**) Significant difference between male and female percent composition determined by one-way ANOVA with Bonferroni’s posttest (p < 0.05).
BECs Urine
94 of 43
Association between iAs in drinkingwater and diabetes
0-25 26-50 51-75 76-1000
2
4
6
8
10
12
14
* * *
PercentileiAs in Drinking Water
OR
Association of diabetes with exposure to iAs in drinking water adjusted for age, sex, and BMI. Diabetes is classified by either FPG
≥ 126 mg/dL, 2HPG ≥ 200 mg/dL, self-report of doctor diagnosis or use of anti-diabetic medication, n = 374. (*) p < 0.05 for the comparison of cases to non-diabetic individuals. The values for each IQR are presented on Slide 78.
95 of 43
Markers of As exposure areassociated with FPG and 2HPG
Associations between log-transformed FGP and 2HPG with iAs in drinking water and iAs metabolites in BECs determined by linear regression adjusted for age, sex, and BMI. Coefficients are standardized to an increment of one inter-quartile range. p-value for test of β = 0.
FPG 2HPGß SE P r2 ß SE p r2
iAs in Water 0.041 0.006 <0.01 0.21 0.031 0.006 <0.01 0.17iAsIII 0.056 0.009 <0.01 0.16 0.042 0.009 <0.01 0.14MAsIII 0.062 0.009 <0.01 0.17 0.050 0.009 <0.01 0.15DMAsIII 0.050 0.011 <0.01 0.12 0.039 0.011 <0.01 0.12iAsV 0.013 0.009 0.15 0.07 0.007 0.009 0.45 0.09MAsV 0.015 0.008 0.08 0.08 0.010 0.008 0.21 0.09DMAsV 0.010 0.009 0.25 0.07 0.007 0.009 0.40 0.09Sum AsIII+V 0.045 0.011 <0.01 0.11 0.035 0.011 <0.01 0.11MAs/iAs 0.046 0.022 0.04 0.08 0.039 0.022 0.08 0.09DMAs/MAs -0.072 0.015 <0.01 0.12 -0.057 0.016 <0.01 0.12DMAs/iAs -0.032 0.013 0.01 0.08 -0.033 0.016 0.04 0.10
BEC
s
96 of 43
Markers of As exposure areassociated with FPG and 2HPG
Associations between log-transformed FGP and 2HPG with iAs in drinking water and iAs metabolites in BECs determined by linear regression adjusted for age, sex, and BMI. Coefficients are standardized to an increment of one inter-quartile range. p-value for test of β = 0.
FPG 2HPGß SE Pc r2 ß SE pc r2
iAs in Water 0.041 0.006 <0.01 0.21 0.031 0.006 <0.01 0.17iAsIII 0.056 0.009 <0.01 0.16 0.042 0.009 <0.01 0.14MAsIII 0.062 0.009 <0.01 0.17 0.050 0.009 <0.01 0.15DMAsIII 0.050 0.011 <0.01 0.12 0.039 0.011 <0.01 0.12iAsV 0.013 0.009 0.15 0.07 0.007 0.009 0.45 0.09MAsV 0.015 0.008 0.08 0.08 0.010 0.008 0.21 0.09DMAsV 0.010 0.009 0.25 0.07 0.007 0.009 0.40 0.09Sum AsIII+V 0.045 0.011 <0.01 0.11 0.035 0.011 <0.01 0.11MAs/iAs 0.046 0.022 0.04 0.08 0.039 0.022 0.08 0.09DMAs/MAs -0.072 0.015 <0.01 0.12 -0.057 0.016 <0.01 0.12DMAs/iAs -0.032 0.013 0.01 0.08 -0.033 0.016 0.04 0.10
BEC
s
97 of 43
Markers of As exposure areassociated with FPG and 2HPG
Associations between log-transformed FGP and 2HPG with iAs metabolites in urine determined by linear regression adjusted for age, sex, and BMI. Coefficients are standardized to an increment of one inter-quartile range. p-value for test of β = 0.
FPG 2HPGß SE P r2 ß SE p r2
iAsIII+V 0.048 0.009 <0.01 0.13 0.043 0.009 <0.01 0.14MAsIII+V 0.062 0.010 <0.01 0.15 0.045 0.011 <0.01 0.13DMAsIII+V 0.078 0.011 <0.01 0.18 0.061 0.011 <0.01 0.15Sum AsIII+V 0.076 0.011 <0.01 0.17 0.059 0.011 <0.01 0.15MAs/iAs -0.001 0.019 0.95 0.02 -0.032 0.019 0.05 0.09DMAs/MAs 0.072 0.036 0.05 0.08 0.100 0.036 <0.01 0.11DMAs/iAs 0.016 0.018 0.37 0.07 -0.005 0.018 0.80 0.09
Urin
e
98 of 43
Markers of As exposure areassociated with FPG and 2HPG
Associations between log-transformed FGP and 2HPG with iAs metabolites in urine determined by linear regression adjusted for age, sex, and BMI. Coefficients are standardized to an increment of one inter-quartile range. p-value for test of β = 0.
FPG 2HPGß SE P r2 ß SE p r2
iAsIII+V 0.048 0.009 <0.01 0.13 0.043 0.009 <0.01 0.14MAsIII+V 0.062 0.010 <0.01 0.15 0.045 0.011 <0.01 0.13DMAsIII+V 0.078 0.011 <0.01 0.18 0.061 0.011 <0.01 0.15Sum AsIII+V 0.076 0.011 <0.01 0.17 0.059 0.011 <0.01 0.15MAs/iAs -0.001 0.019 0.95 0.02 -0.032 0.019 0.05 0.09DMAs/MAs 0.072 0.036 0.05 0.08 0.100 0.036 <0.01 0.11DMAs/iAs 0.016 0.018 0.37 0.07 -0.005 0.018 0.80 0.09
Urin
e
99 of 43
Folate Metabolism
100 of 43
Insulin Signaling and Secretion
Examples of As species identified in biological samples (Francesconi, Suzuki, Creed, Thomas, et al..)
Mammals
(methyl)-As-glutathione complexes
(di,tri)methyl-thio-arsenicals