EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA …acuacultura.espe.edu.ec/wp-content/uploads/2016/06/27... · 2016. 6. 1. · EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA FISIOLOGÍA

EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA FISIOLOGÍA DE LA REPRODUCCIÓN EN PECES

Renata G. Moreira Whitton

Department of Physiology, Biosciences Institute, University of Sao Paulo, Brazil

[email protected]

Effects of pollutants in the physiology of fish

POLLUTANTS Osmoregulation

Metabolism

Endocrine

Biochemical Cellular

Growth Behaviour

Reproduction Vitellogenesis & Steroidogenesis; Substrate deposition; egg development; Brain-Pituitary-Gonads axis alteration; sexual behaviour alteration; genetic damages.

Aluminum

The third element in abundance in the earth's crust;

Brazil is one of the largest producers;

Natural sources: rock erosion;

Anthropogenic sources: industry (metallurgy, textile, pulp and paper, pharmaceutical, manufacturing cosmetics) and water treatment to obtain potable water.

Alumínio bauxite feldspar aluminum

Manganese

• 11º element in abundance in the earth's crust;

• Present in the soil derived from oxides, carbonates, silicates and sulphates;

• The excess of manganese accumulated in the liver and central nervous system resulting from prolonged exposure by inhalation, causes symptoms such as "Parkinson“ disease (degenerative). For these and other adverse effects is that manganese is considered toxic.

Primary effects

• Gills

• Osmoregulation

• Ionoregulation

Secondary effects

• Metabolic

• Endocrine

• Reproduction

Manganese

Gonadal steroidogenesis

Fertility of males

Plasmatic concentration of FSH

and testosterone

Lipoperoxidation and metabolic

stress

Aluminum

Effects on HPG and HPI axis

Alters the concentrations of

sexual hormones (androgens,

estrogens and progestagens)

Acidic pH

Effects on plasma hormonal

concentration

Alters production on sexual

cells

Alterations caused by metals on hepatic vitellogenesis in teleosts

Al/Mn

VTG

PO4

E2

Mn+

RNAm

Mn+

Enz. quinases

lipídios

lipídios

lipídios

lipídios

X

X

X

Al/Mn

Percentages of non-compliant results with respect to the standards established for Class 2, in 2013 and in the period 2008-2012.

Main Basins in Brazil

Southeast region

São Paulo State Basins

1) Considering that aluminum affects fish physiology, can the brain-pituitary-gonads axis be impaired in the presence of this metal?

QUESTIONS RAISED:

3) In vivo and in vitro effects of aluminum are similar?

2) What are the effects of aluminum on the major energetic substrates of vitellogenic females?

4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?

Teleost fish species with different reproductive strategies

Astyanax altiparanae, “lambari” , group synchronous with high

plasticity in the reproductive tatics

Oreochromis niloticus, “tilapia”, asynchronous

Source: MPA

Tilapia and lambari production in Brazil

Production of 180.000.000 fingerlings: -Live bait -Research

(Sussel, 2016)

Experimental Groups:

1) Control (neutral; pH 7.0) (ctr – n) 2) Acidic pH (acidic; pH 5.5) 3) Aluminum (0.5 mg.L-1; pH 7.0) (Al-n) 4) Aluminum (0.5 mg.L-1; pH 5.5) (Al-ac)

1) Considering that aluminum affects fish physiology, can the brain-pituitary-gonads axis be impaired in the presence of this metal?

ACUTE EXPERIMENT – 96 hours

Brain

Neurotransmitters

neurohypophysis adenohypophysis

Gonads

Pituitary stalk

GnRH (+)

GnIH (-)

Dopamine (-)

FSH

Gonadal Steroids

LH

Fee

db

ack

(+

) o

r (-

)

(Medrado, 2014)

Brain-Pituitary-Gonads Axis controlling vertebrate reproduction

Comparing the amount of mRNA copy number FSH normalized by the number of mRNA copies EF1A in the experiment of acute exposure of Oreochromis niloticus to aluminum in neutral pH (7.0) and acid pH (5.5) and respective groups control. Different letters indicate significantly difference among treatments (P <0.05) (Narcizo, 2009).

a a

b

b

Estradiol plasma concentration of mature females of Oreochromis niloticus exposed for 96h to aluminum in water with neutral pH (Al-N) and acid pH (Al-Ac). Also fish were maintained in the absence of aluminum in water with neutral pH (CTR-N) and acid pH (CTR-Ac). Data are expressed as mean ± SEM. Different letters indicate significantly difference among treatments (P <0.05) (Correia et al. 2010).

Aluminum as endocrine disruptor

No effect

-B-P-G axis regulates E2 synthesis -females were already vitellogenic

Oreochromis niloticus

Plasma concentrations of 17α-hydroxyprogesterone of mature females of Oreochromis niloticus exposed for 96h to aluminum in water with neutral pH (Al-N) and acid pH (Al-Ac). Also fish were maintained in the absence of aluminum in water with neutral pH (CTR-N) and acid pH (CTR-Ac). Data are expressed as mean ± SEM. Different letters indicate significantly difference among treatments (P <0.05) (Correia et al. 2010).

Comparing the amount of mRNA copy number LH normalized by the number of mRNA copies EF1A in the experiment of acute exposure of Oreochromis niloticus to aluminum in neutral pH (7.0) and acid pH (5.5) and respective groups control. Different letters indicate significantly difference among treatments (P <0.05) (Narcizo, 2009).

a

b b

a

Aluminum as endocrine disruptor

Impairment in spawning!


Lubzens et al., (2009)

LH

FSH

STEROIDOGENESIS IN FEMALE FISH

Maturation-inducing hormone (MIS)

- Energetic investment to accelerate spawn;

- Due to the mechanism of asynchronic oocyte development, O. niloticus has the physiological plasticity to accelerate the mobilization process of vitellogenesis, resulting in an increased lipid concentration in ovaries.



-Group synchronic species – Hepatic lipid also decreased but it was not mobilized to the ovaries; -Al increased liver lipoperoxidation in mature A. altiparanae


No effects

Astyanax altiparanae



Incubation of pituitaries of O. niloticus in cell culture dish of 24 wells in 200ul of L15 medium in experimental conditions.

-Pituitaries:

Control: only put in L15

GnRH: L15 + GnRH (10um)

GnRH + Al: L15 + GnRH (10um) + AlCl3 (0.1 M)

Al: L15 + AlCl3 (0.1 M) (Hwang et al., 2000)

Analysis of gonadotropins gene expression by real time PCR after 24 hours


-Ovaries: Fractions of 50mg of ovaries were incubated in Krebs-Ringer-HEPES-glucose buffer: Only Krebs: endogenous control Krebs + hCG (6ug/ml) Krebs + hCG (6ug/ml) + AlCl3 (0.1 M) Krebs + AlCl3 (0.1 M)


Analysis of E2 and 17a-OHP after 4 hours

Comparing the amount of mRNA copy number of FSH in vitro experiments from mRNA extracted from pituitary glands of mature female O. niloticus after 24 hours exposure to GnRH 10mM, to GnRH 10mM + AlCl3 (0.1M) and only AlCl3 (0.1M) Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).

ab

a

c bc

Nu

mb

er

of

cop

ies

of

mR

NA

of

FSH

Comparing the amount of mRNA copy number of LH in experiments in vitro from mRNA extracted from pituitary glands of mature female O. niloticus after 24 hours exposure to GnRH 10mM, to GnRH 10mM + AlCl3 (0.1M) and only AlCl3

(0.1M). Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).

ab

a

c

bc

Nu

mb

er

of

cop

ies

of

mR

NA

of

LH


-Al inhibited the cellular response of the pituitary to GnRH; - A similar effect comparing with the in vivo experiment

In vitro effects of Al in tilapia pituitary

a

Concentration of 17αOHP in Krebs-Ringer-HEPES-glucose buffer solution in vitro experiments using fragments of mature ovaries O. niloticus after 4 h of exposure to hCG 6μg/ml; 6μg/ml to hCG and 10 mM Al, and Al only to 10mM. Data are presented as mean ± SEM. Different letters indicate significant differences among treatments (P <0.05).

a a a

b

Concentration of E2 Krebs-Ringer-HEPES-glucose buffer solution in vitro experiments using fragments of mature ovaries O. niloticus after 4 h of exposure to hCG (6μg/ml); to hCG (6μg/ml) and Al (10 mM), and Al only to 10mM. Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).

a a

b

a


-Al inhibited the cellular response of the ovarian steroidogenic cells to gonadotropins; -A similar effect comparing with the in vivo experiment for 17α-OHP but different for E2 (no effect in vivo);

In vitro effects of Al in tilapia ovarian follicles

ab

a

b

0

500

1000

1500

2000

2500

3000

CTRL - neutro CTRL - ácido Alumínio

pg/

mL

17 β ESTRADIOL

AGUDO

Neutral Acidic Aluminum

a

b

0,00

500,00

1000,00

1500,00

2000,00

2500,00

3000,00

CTRL - neutro CTRL - ácido Manganês

pg/

mL

17 β ESTRADIOL

AGUDO

Neutral Acidic Manganese


a

a

b

0,00

500,00

1000,00

1500,00

2000,00

2500,00

3000,00

CTRL - neutro CTRL - ácido Sinérgico

pg/

uL

17 β ESTRADIOL

AGUDO

Neutral Acidic Al + Mn Same effects of Al and Mn – isolated or combined


a

b

0

5

10

15

20

25


pg/

mL

17 α OHP AGUDO


a a

a

b

0,00

5,00

10,00

15,00

20,00

25,00


pg/

mL

17 α OHP AGUDO


a

a

b

0,00

5,00

10,00

15,00

20,00

25,00


pg/

mL

17 α OHP AGUDO

Neutral Acidic Al + Mn

Same effects of Al and Mn – isolated or combined



a a

b

0,00

10,00

20,00

30,00

40,00

50,00

60,00


ug/

dL

Cortisol AGUDO



a a

b

0

10

20

30

40

50

60


ug/

dL

Cortisol AGUDO


0,00

10,00

20,00

30,00

40,00

50,00

60,00


ug/

dL

Cortisol AGUDO

Neutral Acidic Al + Mn Acidic

When Al and Mn are combined, there is no effect




Acidic pH and Al increases androgens

Mn increases E2

Acidic pH increases cortisol

1) Aluminum can be considered an endocrine disruptor in pituitary – ovaries axis, both in

vivo and in vitro, decreasing GtH gene expression and sexual steroids in mature females;

CONCLUSIONS

5) In vitro, Al inhibited the cellular response of the pituitary to GnRH, and the ovarian

steroidogenic cells to gonadotropin, similar effects comparing with the in vivo experiment.

However, the inhibition for E2 synthesis was only demonstrated in vitro;

3) A. altiparanae males use reproductive tactics to trigger changes in testicular

steroidogenesis, by accelerating spermatogenesis and spermiogenesis. On the other hand,

A. altiparanae females prioritized the use of energetic resources during Al exposure

instead of prioritizing reproduction, thereby avoiding the ovulation of impaired eggs.

2) Estradiol levels decrease in group synchronous lambari but does not change in

asynchronous tilapia mature females;

4) The effect of Al and Mn, isolated or combined are not the same;

Thank you!!!!

Documents

EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA …acuacultura.espe.edu.ec/wp-content/uploads/2016/06/27... · 2016. 6. 1. · EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA FISIOLOGÍA