TOXIMED, 2001-2006 - PowerPoint Presentation

Slide1

TOXIMED, 2001-2006

MSc

, 2003

UO, 1995-2005

Graduated

, 2000

UFPR,

2005-2006

IQ-USP, 2006 –

PhD, 2009Post Doctoral, 2010

Unifesp, 2010 –Professor, 2010-LABITA, 2011

TIME OF FORMATION AND EVOLUTION

BIOINORGANIC AND

ENVIRONMENTAL

TOXICOLOGYSlide2

Can

the

manganese to

be related

with these

topics?

Genes Decide Why Some People Love or it

have Music ability and dance preference

http://dx.doi.org/10.3389/fpsyg.2014.00658 http://www.biomedcentral.com/1471-2148/8/210

The molecular basis of color vision in fish and cognitive ability test in this model that can extrapolated for human

The genetic basis of cognition.

http://dx.doi.org/10.1093/brain/122.11.2015

10.1007/s12311-013-0511-x

The Cerebellum's Role in Movement and Cognition

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.

Avdesh

et. al

, 2012;

Ahmad

and

Richardson, 2013

http://

www.nature.com/nature/journal/v496/n7446/full/nature12111.html

Slide3

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

Toxicogenomics

analysis of zebrafish (danio

rerio) embryos reveals pathways

involved in manganese-induced dementiaSlide4

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. Mn

neurotoxicity: 178 years about an unclear historySlide5

Manganese

(

Mn) from earth to human

Human

dietary

requirement for Mn

Upper Intake

Level (UL)

Adequate Intake (AI)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

). Slide6

Mn

in airborne(

MnA)

> drinking water (MnW) for

Mn Neurotoxicity

Authors/Localities

Guideline

MnA

/MnW

Neurologic symptomsBaldwin et al., 1999; Hudnell, 1999

mean (n = 297) women (n = 156)Quebec (Canada)a0.05µg/m

3b0.15µg/m3

MnTP0.009-0.035µg/m3MnPM10

0.007- 0.019 µg/m3

motor deficits and mood disorders, similar to those seen in occupationally exposed workers

Menezes-Filho et al., 200983 children aged 6 - 12 years Bahia (Brazil)

Mn

PM2.5

0.15 µg/m3

poorer cognitive performance,

He et al., 1994

92 children 11–13 years of age from Shanxi (China)

300

μg

/L

a

400

μg

/L

b

1000

μg

/

L

c

240–350 µg/L

impaired manual dexterity and speed, short-term memory, and visual identification…

Wasserman et al., 2006

142 children of 10 years of age from Bangladesh

800 µg/L

lower intelligence quotient (IQ)

Bouchard et al., 2011

362

children

of

6–13

years

of

age

from

Quebec (Canada)

34 µg/L

(1–2,700 µg/L)

MnW

10-fold ↑  2.4 IQ 

Total particulates (TP); PM10 and PM2.5 (particles measuring 10µm, 2.5 µm or less)

aUSEPA, 2004; b Air quality guidelines for Europe, 2000/ bWHO, 2006; cPortaria-MS No 518/2004.Brazil

Despite airborne exposure for

Mn

(>1 mg/m

3

)

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.Slide7

The environmental “safe levels”

for Mn

should be reviewed!, specially in aquatic systems.

In

vitro

In vivo

Hernández

et al., JEM

2009, Hernández

, 2009, Hernández et al., 2015

Environmental Mn

Fractionation

Slide8

Mn

The

Mn

can be

good

and bad too

Parkinsonism

Alzheimer

Autism

<

0.3–2.9 μg /g

> (Aschner & Aschner, 2005

)

ALS

Acromegaly

Catabolic desease

Epilepsy

Chronic

toxicity

is essential for

normal prenatal and neonatal development

...for bone mineralization

, protein and energy

metabolism

,

cellular

protection

from

free

radical

species

,

etc

Deficit

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

Attention Deficit

Hyperactivity Disorder

(ADHD)

Hernández et al., 2011

Hernández et al,

manuscript in eleborationSlide9

What is

Toxicogenomics and which its role in the studies about Mn Neurotoxicity

?

Mn

?

?

?

?

?

?

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.

Differential

expression of

several metal

transport

systems within the

developmental

period

Aschner

et al.

,

2007

Mn disrupted proteins involved in glycolysis, excitotoxicity and cytoskeletal dynamics.Wegrzynowicz et al., 2012Biochemical changes

identified in manganese-exposed

monkeys included endpoints relate to oxidative stress (e.g.,

oxidized

glutathione

)

and

neurotransmission

(

aminobutyrate

,

glutamine

,

phenylalanine

).

Dorman

et al.,

2008

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

i

n

experimental

animals

caused

by

drinking water (DW)

Overexposure

Krishna

et al., 2014Slide10

Objectives

and justification for studies aboutMn Neurotoxicity in vivo using the zebrafish embryo model

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).

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. 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).

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 wild-type zebrafish

embryos model. Slide11

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., 2015Slide12

Manganese-induced

embryotoxicity

depends

on developmental

stage and of the time exposure

Hernández et al, 2015

Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl

2

) and B) comparison of the LC50 of the MnCl

2 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. Slide13

Hernández et al, 2015

Chemical speciation

is important for

manganese-induced

embryotoxicity

Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl

2

) and B) comparison of the LC

50

of the MnCl

2

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 LC

50

from three independent experiments ± standard deviation. Slide14

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. Slide15

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

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 represent means from 3-4 independent experiments ± standard deviation. *** = significant different from each other with respect to the control, H

2O-ISO (T-test, p≤0.001), * = significant different among metal fraction in each 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. Slide16

Mn

(II) but not Mn

(III) induced calcium disruption, which is a plausible cause of Mn-induced

embryotoxicity

Hernández et al, 2015

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 (H2

O-ISO) and 6 mM

citrate, respectively (t-test, p≤0.05).

Mn(II)



Slide17

Hernández et al, 2015 (

unpublished data)

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 resultsSlide18

Hernández et al, 2015 (

unpublished

data)

Mn(II)Cit

is more important than

MnCl2 for

manganese-induced differential gene expressionSlide19

Hernández et al, 2015 (

unpublished data)

Mn

(II)

Cit

is more important than MnCl

2

for manganese-induced differential gene expression

Representative Data

of differential gene expression for one-way

anova

+

pFDR

analysis (significant results to

pFDR < 0.3)Slide20

Hernández et al, 2015 (

unpublished data)

Mn

(II)

Cit

is more important than

MnCl

2

for

manganese-induced

differential gene expression

Representative Data

of differential gene expression for one-way

anova

+

pFDR

analysis (significant

results

to

pFDR

< 0.3)Slide21

Hernández et al, 2015 (

unpublished data)

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).

Chemical

fractionation not appear

to be important for Mn-

induced gene expression in zebrafish

embryosSlide22

qRT

-PCR results

confirmed micro-array findings and the major toxicity of the

Mn(II)Cit

Hernández et al, 2015 (

unpublished

data)

Relative

quantitative

Gene expression., qRT-PCR (bcta2,

cpa1, eif2s1a, mmp2, sgce

e ubqln4

) in

zebrafish

embryos

exposed

from 48-120 hours post

fertilization for

MnCl

2

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

MnCl

2

e

Mn(II)Cit.Slide23

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 METALSlide24

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 disordersSlide25

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 Slide26

Works in course (not showed here) about p

roteomics 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)Slide27

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)Slide28

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.Slide29

Mn

speciation is important for

manganese-induced

neurocognitive impairment (preference or avoid color)

COLOR

PREFERENCE

COLOR

AVOIDE

NEUTRAL

YELLOWBLACK

ORANGEH2

O-ISO Mn(II)Cit

Citrate, MnCl

2

e Mn(III)Cit

AREA

PREFERENCE

CENTRAL

PERIPHERY COLOUR ENRICHED

The pattern of preference color area of zebrafish

embryos,

according to frequency of random visits

of each area. Bars

represent means +

sd

, n=12.Slide30

Mn

speciation is important for

manganese-induced

Neuromotor

(speed) and neurocognitive (

colour

recognizing) impairment

SPEED



NEUTRAL

YELLOW

BLACK

ORANGE

H2

O-ISO Mn(II)Cit

Citrate

, MnCl

2

e Mn(III)Cit

SPEED



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

colour-enriched

enviroment





The zebrafish embryos speed

per

area

for both low and high activity. Bars represent means +

sd

, n=12.Speed (mm/s)Speed (mm/s)Slide31

Conclusions

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).

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 c

an 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).

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.

These results all together can to improve the environmental manganese risk assessment and management. Slide32

TEAMSlide33

ACKNOWLEDGEMENTS

Ministery

of Health,

Spain

Prof. Pannia

Espósito -

team

IQ-USP

, Brazil, 2006-to day

Prof. Scholz

Stefan - teamUFZ, Germany,

2009-to day

Prof. Suñol Cristina - teamIIBB, Spain,

2007-to day

Financing

Collaborators

Thank you very much

by

your attention

Prof.

Diogo

GBB,

UNifesp

,

Brazil,

2012-to

day

Prof. Georgia,

UNifesp

, Brazil,

2012-to day

Prof. Iracilda – Team

Unesp

,

Brazil

2014 -

to

day

Prof. Souza-Pinto

- team

IQ-USP, Brazil,

2009-to day

Prof.

Barbosa Junior- -

team

USP-RP, Brazil,

2010-to

day

Prof. Dr. B. Michalke Team

German Research Center for Environmental

Health,

2012- to day

Prof. Michael Aschner

Vanderbilt

University

,

USA.

2011-to day