RUNNING INTERFERENCE: PHYSICAL ACTIVITY IMPACTS THE GUT-BRAIN AXIS AND BRAIN

METASTASIS INDUCTION BY POLYCHLORINATED BIPHENYLS

Michal Toborek Department of Biochemistry and Molecular Biology

University of Miami School of Medicine

Environmental toxicants with the effects on BBB disruption •Heavy metals (arsenic, cadmium, lead, alumina)

•Nanoparticles, including nanoalumina

•Polycyclic aromatic hydrocarbons (PAHs))

•Diesel exhaust particles

•Nicotine/tobacco smoke

•2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

•Polychlorinated biphenyls (PCBs)

•209 congeners •Toxicity depends upon the number and the positions of chlorines present on the biphenyl structures. •The higher the chlorine content of a PCB, the less the biodegradability.

•More than one million capacitors and 14,000 transformers containing PCBs are still in use in the U.S. ; the total amount of PCBs in registered transformers in the EPA database is 4.7x107kg. •Primary sources of PCB exposure include contact with ground water or soil contaminated due to inappropriate disposal of materials containing PCBs, food contamination from food storage in silos with PCB-coated interiors, and fish consumption from contaminated water.

Red indicates sites currently on the National Priority List, yellow is proposed, green is deleted (usually meaning having been cleaned up).

Zhao et al., Common commercial and consumer products contain activators of the aryl hydrocarbon (dioxin) receptor. PLoS ONE, 2013

paper, rubber, and plastic products

PCB77 (dioxin-like; coplanar): AhR (aryl hydrocarbon receptor)

PCB153 (ortho-substituted; non-coplanar): constitutive andorstane receptor (CAR); pregnane-X receptor (PXR), ryanodine receptors (RyR1 and RyR2), epidermal growth factor receptor (EGFR), toll-like receptor 4

PCB118 (mono-ortho-substituted): mixed receptor mechanisms

Health effects of PCB exposureHealth effects of PCB exposure Cl

Cl Cl

Cl Cl

Cardiovascular diseases

Induction of Phase I and Phase II enzymes

Oxidative damage

Exposure

Cancer and cancer metastases

Dysfunction of reproductive and nervous systems

Increased cell proliferation

Suppression of the immune system

In Vitro Mechanisms

Non-coplanar PCBs

Tumor Metastasis

Inflammation MAPKKK PI3K Angiogenesis

MAPKs Akt

Hyperpermeability Endothelial

VEGF

MMPs ECM degradation Dysfunction

IL-8 Endothelial migration

VCAM, ICAM-1, E-selectin, MCP-1

SFK

EGFR

JAKs

AP-1 NF-B

STATs

*EGFR, Epidermal growth factor receptor; JAK, Janus kinase; SFK, Src family kinase

Claudin-5 Phase Contrast Merge

PCB 118

PCB 153

Vehicle

PCB 126

Claudin-5 (Red); DAPI (Blue)

PCB exposure decreases claudin-5 immunoreactivity in cerebral microvessels. Claudin-5 immunoreactivity in brain capillary microvessels was evaluated using confocal microscopy. The images were acquired using a 60x oil-immersion lens and under identical instrument settings. Claudin-5 immunoreactivity (stained in red) is reflected in the left panel. Additional staining with DAPI was performed to visualize the nuclei (stained in blue). Phase contrast micrographs (middle panel) illustrate the isolated microvessels and merged micrographs (right panel) localize claudin-5 immunoreactivity within the microvessels.

Vehicle PCB 118 PCB 126 PCB 153

ZO-1

Occludin

Merge

Phase contrast

Individual PCB congeners differentially influence the staining intensity and association of occludin and ZO-1 in cerebral microvessels. Occludin (upper panel) and ZO-1 (2nd panel from the top) immunoreactivity within brain microvessels was evaluated using confocal microscopy. Merged micrographs (3rd panel from the top) were obtained by superimposing images of the corresponding optical sections stained for occludin and ZO-1. Phase contrast micrographs (lower panel) localize TJ immunoreactivity within the brain microvessels. Regions of occludin and ZO-1 co-localization are depicted in yellow (arrows).

BB

B P

erm

eabi

lity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

*

*

Vehicle PCB118 PCB126 PCB153

Exposure to PCBs increases the BBB permeability. Animals were treated with PCBs 48 h. Then, 200 μl of 10% sodium fluorescein was injected i.p. and allowed to circulate for 15 min. The ratio of brain to plasma sodium fluorescence level served as a marker of brain permeability.

BRAIN METASTASES

The brain is the major site of cancer metastases and approximately 30-40% of cancer patients develop primary or secondary brain metastases.

~200,000 new cases of brain metastases are diagnosed yearly in the US.

The development of brain metastases is associated with considerable morbidity with median survival of 1-2 months in non-treated patients.

Endothelial cell Cancer cell

BRAIN METASTASES The most common sources of brain metastases are lung cancer (48% of all cases of brain metastases), breast cancer (22-25% of all cases), and melanoma (~15% of cases).

The brain exhibits a compact environment which lacks large extracellular spaces. Therefore, brain metastases which develop in such a restricted space produce early and severe adverse effects.

The brain lacks lymphatic drainage; however, it is highly vascularized. Therefore, the main route of metastatic formation into the brain is hematogenous spread across the BBB.

The BBB protects the brain microenvironment from the majority of the blood-borne components. The disruption of the integrity of the BBB facilitates the entry of tumor cells into the brain.

   

Common carotid artery

Trachea

ECA

ICA

10µm

Tumor cells PCB118+tumor cells

10µm

Control D122-Luc

4.0

3.5

Tum

or C

ell E

xtra

vasa

tion

solitary cells

metastases

3.0

2.5

2.0

1.5

1.0

0.5

Tumor cells PCB118+ Tumor cells PCB118+ Tumor cells Tumor cells

(rel

ativ

e nu

mbe

r)

PCB118+ solitary tumor cells metastases Tumor cells Tumor cells

1.2x106 1000000 Control (vehicle injected mice)Tumor cells

4x105

Tum

or B

iolu

min

esce

nce

PCB118+Tumor cells *1x106 800000

week 2 8x105

600000

6x105 400000

week 4 2x105

200000

0

p/sec/cm^2/sr 0 1 2 3 4 weeks post tumor cell injection

75 mol/kg 150 mol/kg

PCB 118

PCB 126

5x106

4x106

3x106

2x106

1x106

0Bra

in T

umor

Bio

lum

ines

cenc

ePCBs enhance the rate and progression of brain metastasis in a dose-dependent manner. Mice were administered with PCB118 or PCB126 at the dose of 75 or 150 μmol/kg or vehicle (safflower oil) by oral gavage. Forty eight hours later, mice were injected with K1735-luciferase tagged metastatic melanoma cells (5 x 105 cells in 100 μl) in the internal carotid artery. The progression of brain metastases was assessed at day 4 post tumor cell injection.

PCB 75 mol/kg PCB 150 mol/kg

*†

*†

Vehicle PCB118 PCB126

*** ††#ICAM-1 90

**Vehicle PCB118

PCB118+Tumor cells Tumor cells

ICA

M-1

Exp

ress

ion

(r

elat

ive

valu

es) 80

70

60

50

40 Vehicle PCB118 Tumor cells PCB118+

Tumor cells

Vehicle PCB118

PCB118+Tumor cellsTumor cells V

CA

M-1

Exp

ress

ion

(rel

ativ

e va

lues

)

30

40

50

60

70

80

90

100

***

*** †††

VCAM-1

Vehicle PCB118 Tumor cells PCB118+ Tumor cells

•Does oral exposure play a role? Can intestinal factors influence brain metastasis formation upon exposure to environmental chemicals? •The food chain is the main route of exposure to different environmental toxicants, including PCBs. The effects of the majority of environmental toxicants on the gastrointestinal (GI) system are largely unknown.

•We propose that exposure to environmental toxicants through diet may disrupt intestinal epithelium and alter tight junctions (TJs) of intestinal epithelial cells.

•The small intestine is characterized by the presence of villi containing epithelium. The GI epithelium contains complex TJ protein network which effectively limits passive transfer of solutes and ions across the epithelium.

    

Normal barrier Impaired barrier

T cells

T cells

Macrophages

Dendritic cells

Immune activation Inflammation

Inte

stin

al lu

men

Epith

elia

l cel

lsLa

min

a pr

opria

A B

villi

Leukocytes

Apical (absorptive) surface of enterocytes that contains microvilli and negatively charged glycoproteins

Lamina propria

ENMs Enterocytes Goblet Cell

Lumen

Claudins Myosin

Occludin Tight ZO-1F-actin junction

JAM-A MLCK

ZO-1 -catenin

E-cadherin Adherens junction

Tight junction -catenin Desmoglein

Desmosome Keratin

Desmoplakin Desmocolin

Enterocyte

Suzuki, Cell Mol Life Sci. 70, 631, 2013

Orally administered PCBs alter ZO-1 and occludin protein expression in small intestine

60 7 *

Vehicle

PCB 118 Inte

stin

al P

erm

eabi

lity

(rel

ativ

e un

its)

Vehicle PCB118

50

40

30

20

10

Plas

ma

LPS

Leve

ls (n

g/m

l)

***

*** ***

Control PCB153 PCB118 PCB126

6

5

4

3

2

1

0

Cytokine Vehicle

IL-6 (pg/ml) 20.1±3.7

TNF-α (pg/ml) 0.9±0.2

CCL-2 (pg/ml) 25.2±4.7

0

PCB118

55.8±7.9*

2.3±0.6*

33.8±10.7*

PCB 153

Intra-Kingdom Communication Inter-Kingdom Communication Bacteria to Mammalian•Bacteria to Bacteria Human to Bacteria

Important Question Do bacterial factors influence the integrity of the gut epithelium and the brain?

Goal: Understand Bacteria-Mammalian Interactions

Serotonin

Mammalian signaling molecule-microbiome interactions.

Bacterial QSMs-human pathway interactions.

Michal, We are putting it on the web as a pod cast as well and I just wanted to clarify: Is your first name pronounced My-Kal or mee-Kal? With the spelling, I just wanted to make sure! Thanks

Exercise or sedentary Vehicle PCB mixture End of conditions experiment

feces fecesFive weeks of voluntary collection collection exercise or sedentary (2 days) (2 days) conditions

Control data PCB data

Dis

tanc

e (k

m)

16

14

12

10

8

6

4

2

0 0 5 10 15 20 25 30 35

Exercise period (days)

ExercisedExercisedExercisedExercisedExercisedSedentarySedentarySedentarySedentarySedentarySedentary

PCoA1

PC

oA2

-0.2

-0.1

0.0

0.1

0.2

-0.2 -0.1 0.0 0.1 0.2 0.3 -0.02 -0.01 0.00 0.01 0.02

-0.005

-0.010

PCoA1

Exercise alters the structure and composition of the gut microbiome. Bacterial taxa were analyzed in feces by the PhyloChip assay. (A) Principal Coordinate Analysis (PCoA) based on unweighted unifrac distance between exercised and sedentary mice. Opened circle; sedentary mice, closed circle; exercised mice. (B) PCoA and (C) hierarchical clustering-average-neighbor (HC-AN) analysis based on weighted unifrac distance between exercised and sedentary mice of the 2,510 taxa with significant abundance differences across at least one of the categories. Opened circles; sedentary mice, closed circles; exercised mice.

0.010

0.005

PC

oA2

0.000

Sedentary-PCB Sedentary-PCB Sedentary-PCB Sedentary-Vehicle Sedentary-Vehicle Sedentary-Vehicle Exercised-PCB Exercised-Vehicle Exercised-PCB Exercised-PCB Exercised-Vehicle Exercised-Vehicle

Exercise prevents PCB-induced alterations of the gut microbiome. Opened circles, sedentary mice before PCB treatment; closed circles, sedentary mice after PCB treatment; opened squares, exercised mice before PCB treatment; closed squares, exercised mice after PCB treatment.

Table 1. Ten the most decreased bacterial OTUs by PCB mixture in sedentary mice. Phylum Class Order Family Species GenBank Fold

Accession ID Change Proteo bacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas EF645247 -5.6

plecoglossicida str. CGMCC 2093

Proteo bacteria Gammaproteo bacteria Pseudomonadales Pseudomonadaceae FJ901066 -4.8

Proteo bacteria Betaproteo bacteria Burkholderiales Comamonadaceae GQ007353 -4.4

Proteobacteria Betaproteo bacteria Burkholderiales Comamonadaceae GQ108141 -4.4

Proteo bacteria Gammaproteo bacteria Pseudomonadales Pseudomonadaceae Pseudomonas DQ095882 -4.3 plecoglossicida str. R18

Proteobacteria Betaproteo bacteria Burkholderiales Comamonadaceae GQ008724 -4.3

Proteo bacteria Betaproteo bacteria Burkholderiales Comamonadaceae GQ100754 -4.1

Proteobacteria Gammaproteo bacteria Pseudomonadales Pseudomonadaceae Pseudomonas EU826028 -4.0 putida str. SRI156

Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus GQ077246 -4.0 infantis

Proteo bacteria Betaproteo bacteria Burkholderiales Comamonadaceae EF520494 -4.0

Table 3. Prediction analysis for microarrays (PAM)-selected distinctive bacterial species presenting differentially in gut microbiome of exercised and sedenta_!1 mice.

Phylum Class Order Family Species GenBank Fold Accession ID Change

Increased Enterococcus species in Firmicutes Bacilli Lactobacillales Enterococcaceae EF533987 24.1

faecium exercised mice Enterococcus

Firmicutes Bacilli Lactobacillales Enterococcaceae AY692451 15.7 faecium

Staphylococcus Firmicutes Bacilli Bacillales Staphylococcaceae DQ350835 12.1

gallinarum

Escherichia coli Firmicutes Bacilli Lactobacillales Enterococcaceae FJ675223 7.6

0157:H7

Enterococcus Firmicutes Bacilli Lactobacillales Enterococcaceae FJ378658 7.4

faecium

Streptococcus Firmicutes Bacilli Lactobacillales Streptococcaceae GQ000464 8.7

pseudopneumoniae

Bacillus Firmicutes Bacilli Bacillales Bacillaceae AB434284 5.8

trypoxylicola

Decreased species in Erysipel otrichi Erysipelotrichales Erysipelotrichaceae Cll K211 DQ015346 -361

exercised mice

Firmicutes Clostridia Clostridiales Ruminococcaceae EU453081 -6.4

Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides clarus AB490801 -8.6

Exercise helps to maintain the integrity of the BBB

JAM-A ZO-1

Sedentary+Vehicle Sedentary+Tumor Cells Sedentary+Vehicle Sedentary+Tumor Cells

Exercised+Vehicle Exercised+Tumor Cells Exercised+Vehicle Exercised+Tumor Cells

Exercise protects against the development of brain metastases

Sedentary

Exercise

MolecularBiologyBiochemistry

Cell-to-Cell Communication In Gram Negative Bacteria

Chemical Signal Molecules: N-acyl homoserine lactones or AHLs

Sender

Sender

Receiver

Receiver

AHLs

• Synthesis • Secretion/Diffusion • Control • Receptors/Regulators • Sensing • Transduction

• Receptors/Regulators • Sensing • Transduction • Synthesis • Secretion/Diffusion • Control

AHLsO

O

N R

O

“Quorum” is the minimum number of members required to make group-based decision

Mechanism by which bacteria sense their population density, and regulate gene expression depending on population size

Signaling molecules: N-acyl homoserine lactones (AHLs), oligo-peptides, autoinducer 2 (AI-2), and others

N-acylated homoserine lactones (AHLs) •Gram Negative •Can vary in length from 4 to 18 carbon chain. •Detected by various receptors including LasR,

RhlR, and QscR

Autoinducing peptides (AIPs) •Gram Positive

Autoinducer-2 compounds (AI-2s) •Gram Negative and Positive •Detected by LuxP

Known Effects of Microbial QSMs in Mammalian Circuits

AHL

AHL

AHL

             

0

4

C12‐HSL increases permeability of intestinal epithelial cell barrier

3.0

PBSVehicle

10 50 100 200 10 50 100 200

C12-HSL C4-HSL

*

***

Perm

eabi

lity

(% c

ontr

ol)

***

†††

Perm

eabi

lity

(% c

ontr

ol)

2.5

2.0

1.5

1.0

0.5

3

2

1 0.0 PBS Vehicle CSF PCB mix PCB mix

+CSF

C12-HSL (M)

No Veh 50 100 200 400 ZO-1

Occludin

C12-HSL: N-3-oxo-dodecanoyl homoserine lactone C4-HSL: N-butyryl homoserine lactone CSF, competence and sporulation factor (CSF) of the Gram(+) Bacillus subtilis. Bacilli are increased in mice subjected to voluntary exercise.

Tricellulin

Claudin-3

Claudin-15

GAPDH

 

(200 uM)

MMP-2

C12-HSL-induced activation of MMP-2 and MMP-3

C12-HSL (uM)

No Veh 50 100 200 400

MMP-9 (proactive-92kD, active-86kD)

MMP-2 (proactive-72kD) MMP-2 (active-66kD)

MM

P-3

activ

ityM

MP-

2 ac

tivity

(5-F

AM

olig

opep

tide)

(5-F

AM

olig

opep

tide)

(200 uM)

MMP-3 C12-HSL (uM)

No Veh 50 100 200 400

MMP-1 or MMP-3 (proactive-55-57kD)

Casein zymogram

MMP-1 or MMP-3 (active-45kD)

                 

MM

P-3

activ

ity

MMP-3

Relationship between Permeability and MMP activation (F

old

to N

o tr

eate

d)

MMP-2

No Veh ━ M2 M3 GM

MMP-2 and MMP-3 increase permeability through tight junction disruption

MM

P-2

activ

ity

Perm

eabi

lity

(Fol

d to

No

trea

ted)

3O-C12 (200M)

Tricellulin

GAPDH

No Veh ━ M2 M3 GM

Occludin

Perm

eabi

lity

Perm

eabi

lity

(Fol

d to

veh

icle

)

3O-C12-HSL (200 M)

M2: MMP‐2 inhibitor III M3: MMP‐3 inhibitor II GM: GM1489, general MMP inhibitor

               

IL‐8

TNF‐

Induction of intestinal epithelial inflammation by QSMs/role of MMPs TN

Fa m

RN

A e

xpre

ssio

n (F

old

to 0

.5h

cont

rol)

IL-8

mR

NA

exp

ress

ion

(Fol

d to

0.5

h co

ntro

l)

CCL2

IL-8

mR

NA

exp

ress

ion

(Fol

d to

No

trea

ted)

No Veh ━ M2 M3 GM14

IL‐8

CC

L2 m

RN

A e

xpre

ssio

n (F

old

to 0

.5h

cont

rol)

CCL2

3O-C12-HSL (200 M)

CONCLUSIONS

•Both the intestinal epithelium and the brain epithelium are highly susceptible to toxicity of environmental chemicals. Toxicity involves alterations of TJ protein expression and an increase in functional permeability.

•Oral exposure to environmental chemicals directly contributes to the systemic effects of these toxicants, including disruption of the BBB integrity.

•We demonstrate for the first time that physical activity induces a shift in the gut microbiome, favoring the growth of “good” bacteria.

•QSMs, especially long chain long (C12-HSL) AHLs, induce intestinal toxicity and disrupt epithelial integrity.

•The gut-brain axis induced by alterations of the gut permeability has profound impact on the BBB integrity and influences the rate of brain metastasis development and growth.

ACKNOWLEDGMENTSACKNOWLEDGMENTS

Thank

Dr. SY Eum Dr. E. Sipos Dr. YJ. Choi Dr. JJ Choi Dr. M. Seelbach Dr. L. Chen Dr. G. Wolff

Financial Support NIH/NIMH, NIH/NINDS, NIH/NIDA, NIH/NCI NIEHS/Superfund Research Program