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.
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
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
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.