Transcript Hernández et al, 2015 - Toxicogenomics Conferences

BIOINORGANIC AND ENVIRONMENTAL TOXICOLOGY
Unifesp, 2010 –
Professor, 2010LABITA, 2011
IQ-USP, 2006 –
PhD, 2009
Post Doctoral, 2010
UFPR, 2005-2006
TOXIMED, 2001-2006
MSc, 2003
UO, 1995-2005
Graduated, 2000
TIME OF FORMATION AND EVOLUTION
Dementia is classified as a neurocognitive disorder, with various degrees of severity,
that cause a long term and often gradual decrease of cognitive ability and some time
motor ability as well.
The genetic basis of cognition.
http://dx.doi.org/10.1093/brain/122.11.2015
The Cerebellum's Role in Movement and Cognition
10.1007/s12311-013-0511-x
Genes Decide Why Some People Love or it
have Music ability and dance preference
http://dx.doi.org/10.3389/fpsyg.2014.00658
The molecular basis of color vision in fish and cognitive ability test in this model that can extrapolated for human
http://www.biomedcentral.com/1471-2148/8/210
http://www.nature.com/nature/journal/v496/n7446/full/nature12111.html
Avdesh et. al, 2012; Ahmad and Richardson, 2013
Can the manganese to be related with these topics?
Toxicogenomics analysis of zebrafish (danio
rerio) embryos reveals pathways involved in
manganese-induced dementia
Prof. Dr Raúl Bonne Hernández
Bioinorgânica e Toxicologia Ambiental. Departamento de Ciências Exatas e da Terra
Instituto de Ciências Químicas, Farmacêuticas e Ambientais
Universidade Federal de São Paulo
Mn neurotoxicity: 178 years about an unclear history
• Neurological risks associated with occupational exposures is well
established… Couper (1837) was the first that observed and reported the
manganism (Parkinsonian symptoms)
• The environmental guideline have been little studied and its are
based on neurotoxic effects observed in occupationally exposed
workers and unclear animal data (low exposures ≠ elevated
exposures), which suggest more studies for risk assessment of
chronic low-level manganese exposure to humans (Gwiazda et al.,
2007).
• Organisms during development are the more vulnerable for Mn
and the neurotoxicity mechanisms are unclear. Hence, are
necessaries more studies to identify effective biomarkers and to
improve the neurotherapies available.
Manganese (Mn) from earth to human
Mn is a naturally occurring metallic element (#25 in the period table)
throughout the earth’s crust, found naturally in water, air , soil and several
foods, being an essential trace element of metalloproteins for human health,
in different metabolic processes (e.g., gluconeogenesis, energy metabolism,
and antioxidant defenses (Crossgrove and Zheng, 2004).
Human dietary requirement for Mn
Adequate Intake (AI)
Upper Intake Level (UL)
Mn in airborne(MnA) > drinking water (MnW) for Mn Neurotoxicity
Authors/Localities
Baldwin et al., 1999; Hudnell, 1999
mean (n = 297) women (n =
156)Quebec (Canada)
Guideline
MnA/MnW
Neurologic symptoms
0.05µg/m3
b
3
MnTP
0.009-0.035µg/m3
MnPM10
0.007- 0.019 µg/m3
motor deficits and mood
disorders, similar to those seen in
occupationally exposed workers
a
0.15µg/m
Menezes-Filho et al., 2009
83 children aged 6 - 12 years Bahia
(Brazil)
MnPM2.5
0.15 µg/m3
He et al., 1994
92 children 11–13 years of age from
Shanxi (China)
Wasserman et al., 2006
142 children of 10 years of age from
Bangladesh
Bouchard et al., 2011
362 children of 6–13 years of age
from Quebec (Canada)
300 μg/La
400 μg/Lb
1000 μg/Lc
poorer cognitive performance,
240–350 µg/L
impaired manual dexterity and
speed, short-term memory, and
visual identification…
800 µg/L
lower intelligence quotient (IQ)
34 µg/L
(1–2,700 µg/L)
MnW 10-fold ↑  2.4 IQ 
Despite airborne exposure for Mn (>1 mg/m3) is the most important to Mn neurotoxicity,
the increasing number of studies reporting a relationship between Mn exposure for “safe
levels in airborne and drinking water” and children’s health suggests further investigations
about environmental neurotoxicity risk assessment for this metal.
Total particulates (TP); PM10 and PM2.5 (particles measuring 10µm, 2.5 µm or less)
a
USEPA, 2004; b Air quality guidelines for Europe, 2000/ bWHO, 2006; cPortaria-MS No 518/2004.Brazil
The environmental “safe levels” for Mn should
be reviewed!, specially in aquatic systems.
Risk Parameter
level (ppm)
sd
n
LC50-CGC (Neurotoxicity)
LC50-CTX (Neurotoxicity)
Gene-DarT
LC50-DarT (Mortality)
EC50-DarT (Neurotoxicity)
RAC
FII-A
H2O-CETESB
H2O-WHO
LEL
1.9
8,8
186
267
42
76.7
0.08
0.1-0.5
0.4
460
0.6
4
116
117
6
30
0.06
23
12
6
6
6
12
12
Hernández et al., JEM 2009, Hernández, 2009, Hernández et al., 2015
is essential for normal prenatal and
neonatal development...
for bone mineralization, protein and
energy metabolism, cellular
protection from free
radical species, etc
Hernández et al., 2011
Hernández et al, manuscript in eleboration
The brain remain small amount of Mn by long time because it elimination is the lowest of
the whole body, specially during development…  neurotransmitters alteration
(Glutamate, GABA, Dopamine…)  motor and cognitive dysfunction
What is Toxicogenomics and which its role in the
studies about Mn Neurotoxicity?
Neurobehavioral deficits, characterized by locomotor
and emotional perturbations, and nigral glial activation
associated with significant brain Mn deposition are
among the early signs of Mn neurotoxicity in
experimental animals caused by
drinking water (DW)
Overexposure
Krishna et al., 2014
?
Biochemical changes
identified in manganeseexposed monkeys included
endpoints relate to oxidative
stress (e.g., oxidized
glutathione) and
neurotransmission
(aminobutyrate, glutamine,
phenylalanine).
Dorman et al., 2008
?
?
?
?
Mn disrupted proteins involved in glycolysis,
excitotoxicity and cytoskeletal dynamics.
Wegrzynowicz et al., 2012
Results in vitro suggest that neurotoxicity:
is the major problem for Mn exposure, Hernández, 2009
Mn(II) is more toxic than Mn(III) Hernández et al., 2011
Mn is accumulated mainly
in the basal ganglia and the
cerebellum too
Burton et al., 2009;
Fitsanakis et al., 2011;
García et al., 2006;
Sotogaku et al., 2000a;
Yoon et al., 2009.
expression of several metal
? Differential
transport systems within the developmental
period Aschner et al., 2007
Objectives and justification for studies about
Mn Neurotoxicity in vivo using the zebrafish embryo model
 To compare the toxicity of several and representing common aquatic chemical
species of manganese, and
 To determine by toxicogenomics approaches the potential neurotoxicological
mechanism of the manganese and its linking with dementia, using the wildtype zebrafish embryos model.
(1) Fish represent an important species of economic value and are commonly
used as model organism in environmental risk assessment and its embryos
are considered as refinement, if not replacement to experiments with adult
fish and higher vertebrates (Nagel, 2002; EFSA, 2005).
(2) The zebrafish is one of the most popular model species used in genetics,
developmental biology, pharmacology research, and (eco)toxicology providing
a rich source of available information and its embryonic stages appear to
represent the most sensitive life stages for manganese toxicity and to other
chemical too.
(3) Due to the principal similarities among vertebrates the zebrafish embryos
also allow to unravel basic principles of toxicity important for human health
(Eimon and Rubinstein, 2009; Brittijn, et al., 2009).
Toxicological results suggest that the (Mn(II) > Mn(III))
induced significant (p<0.05) lethality and reduced or
altered motility in zebrafish embryos
Hernández, 2009; Hernández et al., 2015
0
Manganese-induced
embryotoxicity
depends
on0
-8
-4 -4
-3
-2
-1
developmental
stage MnCl
and 2of(log
themM)
time exposure
A
a
120
% death of the control
110
100
90
a
80
LC50 (mM)
2-50 hpf
24-72 hpf
24-72 hpf, dc
48-96 hpf
72-120 hpf
48-120 hpf
2-122 hpf
100
70
60
50
50
0
-8
40
b
-4 -4
A
b
0
110
c
100
10
90
d
d
a
80
2-50
24-72
24-72dC
48-96
72-120
LC50 (mM)
B
-1
a
120
0
-2
MnCl2 (log mM)
30
20
-3
70
48-120
2-122
60
Embryo developmental stages-hpf
50
40
b
Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl2) and
of the LC50 of
b B) comparison
30
the MnCl2 in zebrafish embryos at different development stages and with different exposure
durations (dC – chorion manually removed).
c
Hatching occurs between 48 and 72 hpf (hours post fertilization). Treatments that do not20 share a common letter are significantly
different
d
d
10
from each other (p<0.05). Bars represent mean of the LC50 from three independent experiments ± standard deviation.
0
2-50
24-72
24-72dC
Hernández
et al,2-122
2015
48-96 72-120 48-120
Chemical speciation is important for
manganese-induced embryotoxicity
Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl2) and B) comparison of the LC50 of
the MnCl2 in zebrafish embryos at different development stages and with different exposure durations (dC – chorion manually removed).
Hatching occurs between 48 and 72 hpf (hours post fertilization). Treatments that do not share a common letter are significantly different
from each other (p<0.05). Bars represent mean of the LC50 from three independent experiments ± standard deviation.
Hernández et al, 2015
Metallomics results confirmed that both the chemical speciation and the
chemical fractionation are important for manganese-induced embryotoxicity
Schematic representation for chemical fractionation, speciation and total metal analysis ICP, inductively coupled plasma;
OES, optical emission spectrometer; SEC, size exclusion chromatography and MS, mass spectrometry.
Hernández et al, 2015
Contrary to Mn(III), the Mn(II) is more accumulated in the pellet fraction (granule
and membrane component) than the supernatant fraction (stable and denature
proteins, organelles and other cytoplasmic component). Mn appear in tissue majority
as an inorganic specie and trace complexed with citrate in supernatant
Mn Total
Mn Ext
Fe Total
Mn Pellet
Fe Ext
***
400
300
*
200
100
*
mg/kg (dry weight)
mg/kg (dry weight)
80
0
40
20
0
ISO
O-
H2
A
60
Ci
.
t-6
M
0m
C
Mn
mM
1.5
l 2Mn
C
(II)
Mn species
M
.5m
it-1
)C
(III
Mn
M
.5m
it-1
O-
B
H2
ISO
C
.0
it-6
mM
C
Mn
mM
1.5
l2
-1
Cit
(II)
M
.5m
Mn
Mn species
)
)
A) Manganese fractionation in samples from whole zebrafish larvae tissues after exposure to manganese species, from 48 to 120 hpf.
Total (total metal), Ext (metal in liquid extract) and Pellet (non-dissolved metal). Data are shown as g Mn/kg of dry weight. Bars
Cu Total
Cu Ext ± standard Cu
Pellet
Zn Total
represent means from 3-4 independent
experiments
deviation.
*** = significant different from each other
with respect Zn
to Ext
the
40 H2O-ISO (T-test, p≤0.001), * = significant different among metal fraction in each300
control,
treatment (T-test, p≤0.05). B) Representative
SEC-ICP/MS spectra of manganese speciation in samples from whole zebrafish embryos tissues, after exposure from 48 to 120 hpf to
water-ISO (A) and MnCl2 (B). cps = counts per second.
Hernández et al, 2015
Mn
0.
75
m
m
M
M
nC
l2
-1
.5
m
M
M
nC
l2
-3
.0
M
m
n(
M
II)
C
it0.
75
M
m
n(
M
II)
C
it1.
5m
M
n(
M
I I)
C
it3.
M
0m
n(
III
M
)C
it 0.
75
M
m
n(
M
III
)C
it1.
5m
M
M
O
-IS
0
l2
-
nC
M
H
C
it 6.
2O
Mn(II) but not Mn(III) induced calcium disruption, which is
a plausible cause of Mn-induced embryotoxicity
A
(%, m/m, dry weight)
1.5
Na Total
K Total
Mg Total
Ca Total

Mn(II)
1.0

0.5
*
*
*
*
*
it1
)C
M
n(
III
)C
it0
.7
5
.5
m
m
M
M
M
M
n(
III
C
it3.
0m
5m
M
M
n(
II)
C
it1.
M
n(
II)
75
m
C
it0.
-3
.
M
nC
l2
-1
.
l2
M
nC
M
M
0m
M
5m
M
m
-0
.
l2
M
nC
M
n(
II)
B
75
m
.0
it6
C
2O
H
-IS
O
M
0.0
Mn species
Total content of the macronutrients Na, K, Mg, and Ca (B) was analyzed in samples of whole zebrafish larvae tissues after exposure to the
manganese species from 48 to 120 hpf. Data are shown as %, m/m of dry weight. Bars represent means from 3-4 independent experiments ±
standard deviation. () and () denote significantly different from each other with respect to the control (H2O-ISO) and 6 mM citrate,
respectively (t-test, p≤0.05).
Hernández et al, 2015
Transcriptomic results confirmed too that the chemical
speciation is important for manganese-induced embrytoxicity
GENECHIP ZEBRAFISH
TRANSCRIPTOME ARRAY
(AFFYMETRIX)
qRt-PCR: TaqMan® Gene
Expression Assays Protocol
(Applied Biosystems), using
preformulated primers of genes
selected of the microarray results
Hernández et al, 2015 (unpublished data)
Mn(II)Cit is more important than MnCl2 for
manganese-induced differential gene expression
Hernández et al, 2015 (unpublished data)
Mn(II)Cit is more important than MnCl2
for manganese-induced differential gene expression
Probe set
Control
MnCl2 6mM
Fold Change (linear) Anova, p<0.05
pFDR
Gene
Gene name
Dr.4244.1.A1_at
5.82
9.24
-10.64
0.00001
0.154887
cpa1
carboxypeptidase A1 (pancreatic)
Representative
Data
of
differential
gene
expression
for
one-way
anova
+
pFDR
analysis
(significant results to pFDR < 0.3)
Dr.21605.1.S1_at
5.98
8.73
-6.74
0.000022
0.175652
wu:fc25e04
wu:fc25e04
Probe set
Control
Citrato
Fold Change (linear) Anova, p<0.05
pFDR
Gene
Gene name
Dr.3664.1.S1_at
9.7
5.74
15.52
0.000007
0.109028
fundc2
fun14 domain containing 2
Dr.9399.2.S1_at
9.09
5.78
9.93
0.000018
0.141985
ppp2r4
protein phosphatase 2A activator. regulatory subunit 4
Dr.15412.2.S1_at
9.5
5.95
11.71
0.000038
0.198802
tssc1
tumor suppressing subtransferable candidate 1
Dr.14203.1.S1_at
5.42
9.24
-14.12
0.000058
0.22639
ppp1r14aa
protein phosphatase 1. regulatory (inhibitor) subunit 14Aa
Probe set
Citrato
Mn(II)Cit 1.5 mM Fold Change (linear) Anova, p<0.05
pFDR
Gene
Gene name
Dr.14866.1.A1_at
5.16
9.89
-26.55
0.00001
0.159918
gnsa
glucosamine (N-acetyl)-6-sulfatase a
Dr.14481.1.S1_at
9.41
5.87
11.6
0.000072
0.281294
LOC100536058
uncharacterized LOC100536058
Dr.16350.1.A1_at
5.92
10.19
-19.23
0.00005
0.281294
LOC100535214
uncharacterized LOC100535214
Dr.22485.1.A1_at
4.84
9.16
-19.87
0.000067
0.281294
slc14a2
solute carrier family 14 (urea transporter). member 2
Probe set
Citrato
Mn(II)Cit 3 mM Fold Change (linear) Anova, p<0.05
pFDR
Gene
Gene name
Dr.209.1.S1_at
10.02
6.29
13.3
0.000044
0.23421
pitpnbl
phosphatidylinositol transfer protein. beta. like
Dr.8744.1.A1_at
10.54
6.34
18.43
0.000045
0.23421
actr2a
ARP2 actin-related protein 2a homolog (yeast)
Dr.14481.2.S1_x_at
5.55
9.7
-17.84
0.000037
0.23421
LOC100536018
uncharacterized LOC100536018
Dr.2720.1.A1_at
9.8
4.73
33.7
0.000136
0.236234
sgce
sarcoglycan. epsilon
Dr.18285.1.A1_at
5.8
10.34
-23.25
0.000134
0.236234
ell2
elongation factor. RNA polymerase II. 2
Dr.14849.1.S1_at
10.32
4.79
46.18
0.000136
0.236234
myl6
myosin. light chain 6. alkali. smooth muscle and non-muscle
Dr.12375.1.S1_at
5.61
10.19
-23.99
0.000127
0.236234
si:ch211-285f17.1
si:ch211-285f17.1
Dr.399.1.A1_at
10.2
6.97
9.4
0.000134
0.236234
dda1
DET1 and DDB1 associated 1
DrAffx.2.100.A1_at
4.84
9.16
-19.91
0.000122
0.236234
dpp4
Dipeptidyl-peptidase 4
Dr.20032.1.S1_at
9.21
4.91
19.62
0.000247
0.237002
mc5rb
melanocortin 5b receptor
Dr.9964.1.S1_at
5.55
9.59
-16.52
0.000201
0.237002
Dr.3966.1.A1_at
8.83
5.04
13.87
0.000215
0.237002
tagln3b
transgelin 3b
Dr.4325.1.A1_at
9.82
4.88
30.79
0.000246
0.237002
bcat2
branched chain aminotransferase 2. mitochondrial
Dr.1429.1.S1_at
10.29
5.29
31.85
0.000184
0.237002
zgc:193541
zgc:193541
Dr.15634.1.S1_at
5.43
9.85
-21.36
0.000192
0.237002
tcea3
transcription elongation factor A (SII). 3
Dr.16312.1.S1_at
5.5
9.16
-12.61
0.00019
0.237002
sb:cb25
sb:cb25
Dr.14752.1.A1_at
10.11
6.13
15.83
0.000258
0.237002
fam100aa . LOC100536798
family with sequence similarity 100. member Aa ; uncharacterized LOC
Hernández
et al, 2015 (unpublished data)
Dr.26185.1.A1_at
9.14
4.92
18.53
0.000286
0.248387
Mn(II)Cit is more important than MnCl2 for
manganese-induced differential gene expression
Representative Data of differential gene expression for one-way anova + pFDR analysis (significant results to pFDR < 0.3)
Hernández et al, 2015 (unpublished data)
Chemical fractionation not appear to be important for
Mn-induced gene expression in zebrafish embryos
Mn induced equivalent differential gene expression in both the pellet fraction (granule
and membrane component) and the supernatant fraction (stable and denature proteins,
organelles and other cytoplasmic component).
Hernández et al, 2015 (unpublished data)
qRT-PCR results confirmed micro-array findings and
the major toxicity of the Mn(II)Cit
Relative quantitative Gene expression., qRT-PCR (bcta2, cpa1, eif2s1a, mmp2, sgce e ubqln4) in zebrafish embryos exposed from 48-120
hours post fertilization for MnCl2 or Mn(II)Cit em embriões de Danio rerio. The data was normalized to the control water-ISO. Bars
represent (mean ± SD, n = 3).  = p < 0,05 denote significant difference between MnCl2 e Mn(II)Cit.
Hernández et al, 2015 (unpublished data)
Manganese induced calcium homeostasis disruption in zebrafish, followed
of endoplasmic reticulum stress and protein metabolism impairment is a
plausible TOXICOLOGICAL MECHANISMS TO THIS METAL
Hernández et al, 2015 (unpublished data)
Manganese-induced protein metabolism impairment is followed by
catastrophe of several biological process and consequently
neurological diseases, including potential motor and cognitive disorders
Hernández et al, 2015 (unpublished data)
Manganese-induced protein metabolism impairment is
potentially associated with the development of dementia
C
Genes disrupted by Mn-species, which are potentially linked to neurodisorders and dementia, according to
Comparative Toxicogenomics Database. At the same time, it could be biomarkers candidates for early
diagnosis of these pathologies
Hernández et al, 2015 (unpublished data)
Works in course (not showed here) about proteomics studies with
both alternative animal models the zebrafish and the culture of
primary cerebellar granule cells exposed for manganese is
suggesting too that this metal is associated with dementia.
Hernández et al, 2015 (unpublished data)
Phenomics confirmed transcriptomics findings about the
manganese-induced neuromotor and neurocognitive impairment
Esquematic respresentation of neurobehavioral studies with zebrafish embryos of 120 hours post fertilization (hpf), exposed for chemical
species of manganese, from 48 to 120 hpf, using the zebrabox tracking system (Zebrabox/ Viewpoint – França), determining locomotor
activity (distance, time and speed) under environmental complexity (diferente color), allowing to indentfy cognitive impairment too. Analysis
of locomotor activity under neutral color (A) and under diferente color (B) as well as (C) the speed of identification of preference and avoid
of areas colour-enrieched.
Hernández et al, 2015 (unpublished data)
Mn speciation and the environment complexity are important for
Mn-induced neuromotor impairment (time in movement)
Independing of the color environment,
the embryos have the major preference
by freezing activity, and the exposed for
Mn-species have a trend to be stopped
more time in color rich areas, specially for
Mn(III)Cit, during the first 5 minutes.
Total activity (time in movement) of the zebrafish
embryos per area and block time of the experiment.
Bars represent means + sd, n=12.
Mn speciation is important for manganese-induced
neurocognitive impairment (preference or avoid color)
The pattern of preference color area of zebrafish embryos, according to frequency of random visits of each area. Bars
represent means + sd, n=12.
COLOR
PREFERENCE
NEUTRAL
YELLOW
H2O-ISO
Mn(II)Cit
Citrate,
MnCl2 e
Mn(III)Cit
COLOR
AVOIDE
BLACK
ORANGE
AREA
PREFERENCE
CENTRAL
PERIPHERY
COLOUR
ENRICHED
Mn speciation is important for manganese-induced
Neuromotor (speed) and neurocognitive (colour recognizing) impairment
SPEED  
Speed (mm/s)
YELLOW
ORANGE
H2O-ISO
Mn(II)Cit
Speed (mm/s)
Despite zebrafish embryos
have major preference by
freezing activity, when
exposed for species of Mn,
Its have a trend to develop
more speed in the colourenriched enviroment
Citrate,
MnCl2 e
Mn(III)Cit
SPEED  
The zebrafish embryos speed per area for both
low and high activity. Bars represent means + sd,
n=12.
BLACK
NEUTRAL
Conclusions
1) Mn-induced embroy- and neurotoxicity depends on developmental stage,
which is mediated by chemical speciation and chemical fractionation. Indeed,
Mn(II), specially the citrate of Mn(II) appear be more toxic than Mn(III).
2) Mn-induced calcium homeostasis disruption, followed of endoplasmic
reticulum stress and protein metabolism impairment can be an important
mechanism of neuro(toxicity) for this metal, which can be provoking
aggregation/accumulation of proteins and finally dementia (neurocognitive
and neuromotor impairment), which was verified by phenomics approach (it
can be extended for neurobehavioral analysis in human).
3) Toxicogenomics analysis of zebrafish (danio rerio) embryos revealing pathways
involved in manganese-induced dementia suggest that these pathologies can
be appear in young populations exposed for manganese in chronic manner,
and consequently the methods of diagnostic of dementia must be developed
and/or to improved for early life stages too.
4) These results all together can to improve the environmental manganese risk
assessment and management.
TEAM
Thank you very much by your attention
Collaborators
Financing
Prof. Souza-Pinto - team
IQ-USP, Brazil, 2009-to day
Prof. Barbosa Junior- - team
USP-RP, Brazil, 2010-to day
Prof. Pannia Espósito - team
IQ-USP, Brazil, 2006-to day
Prof. Georgia,
UNifesp, Brazil,
2012-to day
Ministery
of Health,
Spain
Prof. Suñol Cristina - team
IIBB, Spain, 2007-to day
Prof. Iracilda – Team
Unesp, Brazil
2014 -to day
Prof. Michael Aschner
Vanderbilt University,
USA. 2011-to day
Prof. Diogo
GBB, UNifesp, Brazil,
2012-to day
Prof. Dr. B. Michalke Team
German Research Center for
Environmental Health, 2012- to day
Prof. Scholz Stefan - team
UFZ, Germany, 2009-to day