Microbiome determinants of

arsenic toxicity

DELAWARE VALLEY DRUG METABOLISM DISCUSSION GROUP

9/17/2019

SETH WALK

MONTANA STATE UNIVERSITY

Michael Coryell

Nicholas Pinkham

Qian Wang Barbara Roggenbeck

Susan Broadaway

Mark McAlpine

Arsenic and the microbiome?

Timothy McDermott

Acutely toxic

Arsenic: a global crisis

200 million people are exposed to harmful levels in drinking water

Group 1 carcinogen

Skin, bladder, and lung cancers

Cardiovascular disease

Hypertension, heart attack, stroke, etc.

Metabolic disorders

Diabetes and obesity

Nervous system dysfunction

Cognitive function and neuropathy

Developmental outcomes

Pregnancy complications, cognitive delays

Arsenic: a global crisis

Agency for Toxic Substance and

Disease Registry

Ranks toxic substances based on:

Frequency

Toxicity

Potential for human exposure

Ranked #1 since 1997

Safe levels?

10 parts per billion (ppb) in human drinking water

Well water

Arsenic metabolism Mobility, accumulation,

and toxicity of arsenic

depend on its chemical

state

Arsenic cycling includes

reductions, oxidations,

methylations, and

thiolations

Trivalent more toxic than

pentavalent

Inorganic more toxic

than organic Cohen et al. Crit Rev Toxicol. 2013. 43(9):711-52

Arsenic (+3 oxidation state)

methyltransferase – AS3MT

Drobna et al. Chem Res Toxicol. 2009. 22(10):1713-20

Liver Urine

Carcinogenicity model

Cohen et al. Crit Rev Toxicol. 2013. 43(9):711-52

What was known

In nearly all environments where arsenic is found, microorganisms

contribute significantly to its mobility and toxicity

McDermott and many others

Arsenic was shown to perturb the gut microbiome of mice

Lu et al. Environ Health Perspect. 2014. Mar. 122(3):284-91.

Gut bacteria were shown to metabolize arsenic in vitro and there

was compelling evidence that this was relevant in vivo.

Van de Wiele et al. Environ Health Perspect. 2010. Jul. 118(7):1004-9.

Dheer et al. Toxicol Appl Pharmacol. 2015. 289:397-408.

Knowledge gap

1) Is microbiome arsenic metabolism beneficial or deleterious to the

host?

(Photo Credit: Giri AK, IICB, India)

Risk factors

Knowledge gap

1) Is microbiome-arsenic interaction beneficial or deleterious to the

host?

2) Does inter-individual variability in microbiome composition

influence disease?

3 orders of

magnitude

Cefoperazone

Antonopoulos et al. Infect Immun. 2009. Jun.

77(6):2367-75.

Coryell et al. Nat Commun. 2018. Dec 21;9(1):5424.

Coryell et al. Nat Commun. 2018. Dec 21;9(1):5424.

3 orders of

magnitude

Cefoperazone

Antonopoulos et al. Infect Immun. 2009. Jun.

77(6):2367-75.

Arsenic (+3 oxidation state)

methyltransferase – AS3MT

Drobna et al. Chem Res Toxicol. 2009. 22(10):1713-20

Liver Urine

Arnold et al. Toxicol Pathol. 2014. Jul. 42(5):855-62

AS3MT-KO mice are

exquisitely sensitive

to arsenic toxicity

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Is a human microbiome sufficient

for protection?

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Knowledge gap

1) Is microbiome arsenic metabolism beneficial or deleterious to the

host?

HIGHLY BENEFICIAL

2) Does inter-individual variability in microbiome composition

influence disease?

Is a human microbiome sufficient

for protection?

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Jonathan Martinson

Martinson et al. ISME J. 2019. Sep;13(9):2306-18.

Does variability in the human

microbiome correlate with disease

outcome?

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Knowledge gap

1) Is microbiome arsenic metabolism beneficial or deleterious to the

host?

HIGHLY BENEFICIAL

2) Does inter-individual variability in microbiome composition

influence disease?

YES

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Highly prevalent genus in human

microbiome samples

Ferments fiber

Butyrate producer

Anti-inflammatory activity

Strictly anaerobic

Used as a probiotic supplement

Faecalibacterium prausnitzii

F. prausnitzii requires a metabolic

partner in the murine gut

Miquel et al. Mbio. 2013. Apr;6(2):e00300-15.

Coryell M et al. Nat Commun. 2018. Dec 21;9(1):5424.

Summary

Arsenic metabolism in the mammalian gut provides protection

during exposure.

Inter-individual variability in microbiome composition results in

differences in protection.

The effect of individual bacteria is detectible in the hyper-sensitive

AS3MT-KO mouse model.

Coryell et al. Nat Commun. 2018. Dec 21;9(1):5424.

Qian et al. Environ Microbiol. 2015. 17(6):1926-40.

Knowledge gap

3) Does bioaccumulation of arsenic decrease toxicity?

MAGTGCKIWEDCKCGAACSCGDSCTCGTVKKGTTSRAGAGCPCGPKCKCTGQGSCNC

VKDDCCGCGK

16 cysteine residues for possible arsenic interaction

Escherichia coli AW3110 and AW3110::fmt

LB broth containing 0, 10, 25, 35, and 50 µM As(III)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10 15

Ce

ll c

on

ce

ntr

atio

ns

(OD

595)

Time (h)

No As

AW3110

AW3110::fmt

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12

Ce

ll c

on

ce

ntr

atio

ns

(OD

595)

Time (h)

10 µM As(III)

AW3110

AW3110::fmt

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15

Ce

ll c

on

ce

ntr

atio

ns

(OD

595)

Time (h)

25 µM As(III)

AW3110

AW3110::fmt

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 5 10 15

Ce

ll c

on

ce

ntr

atio

ns

(OD

595)

Time (h)

35 µM As(III)

AW3110

AW3110::fmt

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15C

ell c

on

ce

ntr

atio

ns

(OD

595)

Time (h)

50 µM As(III)

AW3110

AW3110::fmt

Day 14 (…too early to tell but…)

Mice colonized with fmt-expressing E. coli

No signs of toxicity

Mice colonized with wild-type E. coli

All showing significant precursors to mortality (squinting, hutched

posture, lack of preening, lack of movement, slowing of fecal output)

Knowledge gap

3) Does bioaccumulation of arsenic decrease toxicity?

All signs pointing toward YES

PSA: clonal microbiome dynamics

are important

Jonathan Martinson

Martinson et al. ISME J. 2019. Sep;13(9):2306-18.

2013. Science. 341(6141):1237439

2013

Temporal microbiome dynamics in

healthy individuals are understudied Few studies have been performed

Most rely exclusively on sequencing

Core microbiome persists for

decades

Radio-opque pellets

2.3 days (0.7-4.0)

Resident = present > 14 days

Gap ≤ 30 days

Days per Always

Participant Sample OTUs Resident Resident (Ave)

1 9.5 220 196 (89%) 76 (35%)

2 8.0 244 206 (84%) 99 (41%)

3 17.8 189 179 (95%) 80 (42%)

4 16.6 186 163 (88%) 57 (31%)

5 10.9 179 158 (88%) 77 (43%)

6 8.6 181 125 (69%) 63 (35%)

7 9.5 242 213 (88%) 90 (37%)

8 10.2 181 158 (87%) 94 (52%)

Average 10.5 203 175 (86%) 80 (39%)

All pairwise combinations

within a participant’s

samples:

Significant Positive

Correlation (Pearson) in

all except Participant 2

—However—

Weak Correlation

Coefficient

(range: 0.056–0.377)

&

Small Effect Size of Beta

Diversity Change

(range: 0.04–0.13)

Does microbiome diversity increase over time?

Through the lens of 16S rRNA sequencing, microbiomes sampled close in time

were

nearly as similar as those sampled over long periods of time.

Jonathan Martinson

Martinson et al. ISME J. 2019. Sep;13(9):2306-18.

Enterobacteriaceae >250 species

The most taxonomically diverse bacterial family

All taxa observed in humans can grow on MacConkey agar

Of the 32,470 isolates evaluated

29,306 were E. coli (90%)

3,164 were non-E. coli (10%)

E. coli belonged to at least 120 distinct clones

37 were resident (31%)

3 residents were non-E. coli sensu stricto (2.5%); one of these was a

cryptic Escherichia

E. coli sensu stricto

Non-E. coli sensu stricto

Present for 14

days or more?

Potential Resident

Transient

Yes No

Resident clone

MacConkey Agar

Martinson JV et al. ISMEJ. 2019. May 14. ePub Ahead of Print

Phylogroup Dynamics Within Participant 1

Repetitive elements targeted which

are change rapidly on an evolutionary

timescale.

Summary

Based on 16S sequencing, some OTUs are incredibly stable, but

clones within OTUs change a lot

Does clonal diversity really matter?

Welch RA et al. PNAS. 2002. Dec 24. 99(26):17020-24.

Acknowledgements

Walk Lab

Sue Broadaway

Jonathan Martinson

Nicholas Pinkham

Garrett Peters

Mark McAlpine

Michael Coryell

Barbara Roggenbeck

Qian Wang

Genevieve Coe

McDermott Lab (MSU)

Tim McDermott

Collaborators

France Lefcort – MSU

Valèrie Copiè – MSU

Edward Schmidt – MSU

Brian Bothner – MSU

Ping Li – Chinese U of Geosciences

Lora Arnold – U Nebraska

Samuel Cohen – U Nebraska

X. Chris Le – U Alberta

Masafumi Yoshinaga – Florida International University Animal Resource Center

(MSU)

DuBois Lab (MSU)

Jennifer DuBois

Rand Lab (U Rochester)

Matthew Rand

Funding

Martinson JV et al. ISMEJ. 2019. May 14. ePub Ahead of Print

Human equivalent dose (HED)

Mouse Km = 3, Human Km = 37

Doses typically used to study ACUTE toxicity

10 ppm 811 ppb in humans

25 ppm 2,027 ppb in humans

100 ppm 8,108 ppb

𝐻𝐸𝐷 𝑚𝑔

𝑘𝑔= 𝐴𝑛𝑖𝑚𝑎𝑙 𝑑𝑜𝑠𝑒

𝑚𝑔

𝑘𝑔𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑖𝑒𝑑 𝑏𝑦

𝐴𝑛𝑖𝑚𝑎𝑙 𝐾𝑚

𝐻𝑢𝑚𝑎𝑛 𝐾𝑚

Surface soils

Photo by Stewart Jennings

8% of 5,023 wells were at or

above 10 ppb