Novel Biomarkers for the Risk Assessment of Exposure to Arsenic in Drinking Water

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

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Overview

Introduction Hypothesis Results

Method optimization and validation Population-based study

Conclusions Questions

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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

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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

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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)

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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

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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

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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.

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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

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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

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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

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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.

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Dissertation Hypothesis

Trivalent arsenicals can be quantified in biological systems and used as predictors of

the susceptibility to the diabetogenic effects of As exposure

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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

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Overview




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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

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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

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- + - + - + - +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

*

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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

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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%)

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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%)

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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

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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

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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

* **


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


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

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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

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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.

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Conclusion

It is feasible to design studies systematically analyzing AsIII metabolites in laboratory- and population-based samples.

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Overview




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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

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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

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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

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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

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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)

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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

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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

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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

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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.

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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

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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.

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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.

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Overview




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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.

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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

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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.

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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.

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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.

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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

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Specific Aim 1: Extra Slides

1

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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

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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

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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

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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

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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

* *


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

*

**

*


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

* * *


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

**


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

* *


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


+ +-

MAs,

ng/

reac

tion

0

5

10

15

20a

b,#

a,#b


+ +-

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


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


*

* 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


*

* **

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


*

* *

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


*

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


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


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


*

*

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


Kidney

*

As, n

g/g

tissu

e

0 15 20 25 30 500

500

1,000

1,500

2,000

As3mt-KO WT


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


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


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

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

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

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

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

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

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

Urin

e(n

g/m

L)

-3 -2 -1 0 1 2 3-4

-2

0

2

4

= .41, r2 = .64


Log

MAs

Urin

e(n

g/m

L)

-3 -2 -1 0 1 2 3-4

-2

0

2

4

= .39, r2 = .67


Log

DMAs

Urin

e(n

g/m

L)

-3 -2 -1 0 1 2 3-4

-2

0

2

4

= .40, r2 = .68


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.

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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.

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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

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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

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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.

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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


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


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.


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


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.


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

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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

Date post:	11-Feb-2017
Category:	Science
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