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Can fish liver melanomacrophages be modulated by

xenoestrogenic and xenoandrogenic pollutants?

Experimental studies on the influences of temperature,

sex, and ethynylestradiol, using the platyfish as the

model organism

Carlos Miguel Henriques Ferreira

Dissertação de Mestrado em Contaminação e Toxicologia Ambiental

2011

Carlos Miguel Henriques Ferreira

Can fish liver melanomacrophages be modulated by

xenoestrogenic and xenoandrogenic pollutants? Experimental

studies on the influences of temperature, sex, and

ethynylestradiol, using the platyfish as the model organism

Dissertação de Candidatura ao grau de Mestre

em Contaminação e Toxicologia Ambiental

submetida ao Instituto de Ciências Biomédicas

de Abel Salazar e à Faculdade de Ciências, da

Universidade do Porto

Orientador – Eduardo Jorge Sousa da Rocha

Categoria – Professor Catedrático

Afiliação – ICBAS, Instituto de Ciências

Biomédicas Abel Salazar da Universidade do

Porto; CIIMAR, Centro Interdisciplinar de

Investigação Marinha e Ambiental, Porto

Co-orientador – Maria João Tomé da Rocha

Categoria – Professora Associada

Afiliação – ISCS – N, Instituto Superior de

Ciências da Saúde, Norte; ICBAS, Instituto de

Ciências Biomédicas Abel Salazar da

Universidade do Porto; CIIMAR, Centro

Interdisciplinar de Investigação Marinha, Porto

To all who wish me good and supported me all the way,

specially my family.

1

Acknowledgment

I could not do this work on my own. I have the aid of the most qualified and pleasant

staff to help me do this work and guide me though this work. I could leave a warm thank

you, altogether, but do deserve an individual salute for the amazing they were. So,

For their emotional and moral support, for being present on the good and bad times,

to be available for my demands and for their comprehension, a very special thank you to

my family and to my girlfriend, Sónia;

For all the good times shared, while working or in leisure, for being available

towards work and personal affairs and for his friendship, a special thank you to Francisco

Figueiredo;

For all the good times shared, while working or in leisure, for being available

towards work and personal affairs, and for her friendship, a special thank you to Vera

Sousa;

For all the technical work and support on my work performed on their interships, and

for all the good moments spent with them, a special thank you to Ana Silva, to Sara

Pereira, to Rui Santos and to Catarina Pinheiro;

For her help on various subjects and for taking care of our fishes in particular and for

all knowledge she shared, a special thank you to Catarina Cruzeiro;

For all the support given at the laboratory, for the sharing of her knowledge on the

more histological areas, for their kindness and joy, a very special thank you to Maria

Helena Galante Correia;

For all the support given at the laboratory, for the sharing of her knowledge on the

more diverse areas, for their kindness and joy, a very special thank you to Fernanda

Malhão;

For their support, their readiness to help and support, for sharing their knowledge to

help me end up my work, for their valuable contributions on my work, for the opportunity

they gave me to complete this important step on my professional life and for stimulating

my critical spirit and my interest on the scientific areas, a very special thank you to

Professor Maria João Rocha, and to Professor Eduardo Rocha.

Finally, we want to acknowledge the support of project PTDC/CVT/115618/2009,

financed by national funds via Fundação para a Ciência e a Tecnologia (FCT) / MCTES

(PIDDAC), and co-financed by ―Fundo Europeu de Desenvolvimento Regional‖ (FEDER),

via the COMPETE – ―Programa Operacional Factores de Competitividade‖ (POFC).

―If I have seen a little further it is by standing on the shoulders of Giants.” – Sir Isaac

Newton

2

Index Acknowledgment .............................................................................................................. 1

Figure index ...................................................................................................................... 3

Table index ....................................................................................................................... 4

Abbreviations list ............................................................................................................... 5

Abstract ............................................................................................................................ 7

Resumo ............................................................................................................................ 7

Introduction ......................................................................................................................10

Material and methods ......................................................................................................18

Establishment of better fixative solution ........................................................................18

Establishment of the best staining technique for image analysis ..................................18

Deparaffinization and mounting of sections ...............................................................18

Hematoxylin and eosin ..............................................................................................19

Masson-Fontana .......................................................................................................19

Perl’s prussian blue ..................................................................................................19

Periodic acid Schiff reaction ......................................................................................20

Establishment of baseline sexual and gonadal maturation influences ..........................20

Sub-chronic exposure to a xenoestrogenic compound .................................................20

Tissue sampling, processing, and analyses ..............................................................21

Enzymatic histochemistry assays .............................................................................22

Stereological and histoenzymatic analysis ................................................................22

Individual PMA volume-weighted volume measurement ...........................................23

Immunohistochemistry staining for vitellogenin .........................................................24

Statistical analysis ....................................................................................................25

Results and discussion ....................................................................................................26

Establishment of best staining and fixative solutions ....................................................26

Establishment of baseline sexual and gonadal maturation influences ..........................29

Enzymatic histochemistry assay ...............................................................................40

Exposure to ethynylestradiol ........................................................................................41

Enzymatic histochemistry assay ...............................................................................46

Immunhistochemistry assay ......................................................................................47

Eosinophilia of cytoplasm .........................................................................................47

Conclusion .......................................................................................................................49

Bibliography .....................................................................................................................50

3

Figure index

Figure 1 Diagram demonstrating the hemolytic and oxidative damage that Craft mill

effluents has and how they affect the PMAs density on fish. ............................................13

Figure 2 Diagram about the sexual regulation on fish. Any change on any of the pathways

will usually result on a increase or decrease on the sexual related developments. GhT:

gonadotropins (GhT I = Teleost FSH; GhT II = Teleost LH). ............................................15

Figure 3 Example on how the volume-weighted volume was calculated. The dark lines

with the small grey slashes make the sample grid overlay. Whenever a PMA touches the

grey slash, the length of the dark line (the intercept) is measured (pink on figure) and l is

hence derived to compute the volume. Liver section stained with PAS. ...........................24

Figure 4 a: Male liver stained with H&E. Two PMAs are visible (thicker arrows), next to

blood vessels (star), Lipofuscin pigment is visible even at low magnification (thin arrow) b:

Male liver section stained with H&E. Two PMAs (thicker arrows) presenting well defined

lipofuscin pigments (thin arrows) are spotted next to blood vessels (star). Images c and d:

Male liver sections stained with Masson-Fontana. A small PMA is visible at the thicker

arrow. Thin arrow indicates small melanin pigments. Star: Blood vessel. .........................26

Figure 5 a: Liver section stained with Perl’s prussian blue. One PMAs are visible (thicker

arrows) next to vessels (star) Hemosiderin pigment is visible stained blue (thin arrow). b:

Liver section section stained with Pears. PMAs visible at thicker arrow. Hemosiderin

faintly stained at thinner arrow. c: Liver section stained with PAS. A PMAs is visible at

thicker arrow. Thin arrow indicates several lipofuscin pigments stained. Star: broken tissue

where vessel was. d: Large PMAs stained with PAS where are visible, due to

fragmentation, several macrophages and they lipofuscin content (thin arrows). Star: space

due to tissue shrinkage and blood vessel. ........................................................................27

Figure 6 Liver fixed with 10% formalin. a: PMAs are shown with arrows at very low

magnification. Star shows a zone of broken tissue. H&E. b: Zooming of the area displayed

in a, where it can be seen the relatively poor preservation. A PMA with melanin is pointed

by an arrow. H&E. ............................................................................................................28

Figure 7 Liver fixed with Davidson’s solution. a: The shrinkage effect is pronounced and

visible in all the section. PMAs indicated by arrows, despite hardly seen at low

magnification. H&E. b: A PMA is better visible, intercepted by an artifactual break (arrow).

H&E .................................................................................................................................28

Figure 8 a: liver fixed with 4% paraformaldehyde. An overall good preservation was

attained. PMAs can be spotted in and outside the liver (arrows). b: Zoomed in PMA

showing some degradation on PMA structure. H&E .........................................................29

Figure 9 Liver fixed with Bouin’s solution. a: Preservation is consistently of good quality.

Arrows point to some PMAs, discernible at low magnification. H&E. b: Zoomed in PMA.

PMAs shows to be intact (arrow) H&E .............................................................................29

Figure 10 Graphic chart of gonad weight vs. experimental group. Two different statistical

settings were found (a and b; p<0.05). On set a we can find males together with the high

temperature female, while in set b we can only find females. Box plot graphic with

minimum, first quartile, median, third quartile and maximum. Open circles represent

statistical outliers (herein not excluded from the analysis). ...............................................32

4

Figure 11 Graphic chart of liver weight vs. experimental group. Two different statistical

settings were found (a and b; p<0.05). On set a we can find males together with the high

temperature female, while in set b we can only find females. Box plot graphic with

minimum, first quartile, median, third quartile and maximum. Open circles represent


Figure 12 Graphic chart of relative PMA volume vs. experimental group. Two different

statistical settings were found (a and b; p<0.05). On set a we can find females together

with the high temperature males, while in set b we can only find females. Box plot graphic

with minimum, first quartile, median, third quartile and maximum. Open circles represent


Figure 13 Graphic chart of the absolute volume of PMA vs. experimental group. Three

different statistical settings were found (a, b and c; p<0.05). On set a we can find females

together while in set c we can only find males. In set b we can find a pair, low temperature

female and high temperature male. Box plot graphic with minimum, first quartile, median,

third quartile and maximum. Open circles represent statistical outliers (herein not excluded

from the analysis). ...........................................................................................................38

Figure 14 Graphic chart of volume-weighted volume of PMAs vs. experimental group. Two

statistically significant different sets were found. In set a we can find the females, while in

set b we can find the males. Box plot graphic with minimum, first quartile, median, third

quartile and maximum. Open circles represent statistical outliers (herein not excluded

from the analysis). ...........................................................................................................39

Figure 15 Liver sections histochemically stained for AP. On a and b we can actually see

how the low temperature female (b) has more color saturation than the low temperature

male (a). On the other hand, the high temperature male (c) presents more saturation than

the same temperature female (d). Arrows point to PMAs. ................................................41

Figure 16 Liver sections stained with AP specific enzymatic staining. On a we can see a

liver section of a solvent control animal. In b we can see a liver section of an exposed

animal. Note the more vivid staining at PMA in a (arrow) when compared with that in b. .46

Figure 17 Liver slides stained for vitellogenin, with specific immunohistochemistry. a is a

positive control to the method, using zebrafish liver. b is a liver section of a solvent control

platyfish. c is a liver section of an exposed platyfish. As seen in a, the method did work,

has the hepatocyte cytoplasm is positive for vtg. In the platyfish liver (b and c), the

staining was negative apart from some background staining. ..........................................48

Figure 18 Liver section stained with H&E. In a we have a solvent control animal, with no

exposure to EE2. On b we have an exposed animal which was exposed to 75ng/L of EE2.

Note the difference in the cytoplasm at b, with the cells presenting a more heavily (more

purple) stained on the exposed animal. ...........................................................................48

Table index

Table 1 Demonstrative chart of which enzymes are present in each type of cells of the

immune system. AP: Acid Phosphatase; NBE: α-Naphthyl Butyrate Esterase; BG: β-

Glucuronidase..................................................................................................................16

5

Table 2 Pregnancy stages on females sampled: (-) Not pregnant; (+) Early pregnancy;

(++) Middle pregnancy; (+++) Late pregnancy. ................................................................30

Table 3 Body and organ related gross measurements of fish acclimated to low

temperature .....................................................................................................................31

Table 4 Body and organ related gross measurements of fish acclimated to high

temperature .....................................................................................................................31

Table 5 Liver volume, and relative and total volumes of PMAs of fish acclimated to low

and high temperatures. ....................................................................................................36

Table 6 Volume-weighted volume of PMAs in fish acclimated to low and high

temperatures. ..................................................................................................................38

Table 7 Color saturation of PMAs histochemically stained for acid phosphatase. ............40

Table 8 Body, liver and gonad related gross measurements of control male fish from the

ethynylestradiol assay ......................................................................................................43

Table 9 Body, liver and gonad related gross measurements of exposed male fish from the

ethynylestradiol assay ......................................................................................................43

Table 10 HSI and GSI data and statistical test related to fish exposed ............................43

Table 11 Table with values estimated for liver volume, relative PMA volume and absolute

PMA volume. ...................................................................................................................44

Table 12 Data for the volume-weighted volume of PMAs in the fish analyzed from the

second assay. Each value of a fish represents the mean obtained for that animal. ..........45

Table 13 Data for the collected from volume-weighted volume PMAs in the fish analyzed

from the second assay. Each value of a fish represents the median obtained for that

animal. .............................................................................................................................45

Table 14 Color saturation of AP staining in PM, from fish that experienced the second

assay. ..............................................................................................................................46

Abbreviations list

AP Acid Phosphatase

BG β-Glucuronidase

Cyp Citocrome P-450

EDC Endocrine Disruptor Compound

EE2 Ethynylestradiol

GhT Gonadotropins

6

GSI Gonodosomatic Index

H&E Hematoxylin and Eosin

HSI Hepatosomatic Index

NBE α-Naphthyl Butyrate Esterase

OCT Optimal Cutting Temperature Compound

PAS Periodic Acid Schiff Reaction

PBS Phosphate Buffer Solution

PCB PolyChlorinated Biphenyl

PMAs Pigmented Macrophage Aggregate

RER Rough Endoplasmatic Reticulum

TCDD 2.3.7.8-tetrachlorodibenzo-p-dioxin

Vtg Vitellogenin

7

Abstract

Pigmented macrophage aggregates (PMAs) are distinct groups of pigmented phagocytic

cells present in the liver, kidney and spleen of fish, amphibians and reptiles. They have

been reported to be modulated by endogenous factors as well as by exogenous inputs,

including pollutants. For that reason, some experts have been insisting that PMAs could

be used as biomarkers for environmental stress, at least for some pollutants as in certain

cases they seem not to fit, because of their high variability and poorly known functionality.

Some published data suggests that PMAs may be influenced by sex-steroid influences, at

least in fish, but the issue it still poorly studied. Logically, if such modelling exists toxicants

that mimic estrogenic and/or androgenic effects can act as disruptor of PMAs. To start a

systematic tackle of such questions we selected a sexually dimorphic small fish model,

the platyfish. As a first step we wanted to set an unbiased stereological approach to

quantitatively evaluate changes in the size and liver content of PMAs. Using this method,

we then wanted to study the effects of both sex and temperature (breeding optimal versus

lower non-optimal) on the baseline liver amount and activity of PMAs. Finally, we wanted

to correlate such data with a first assay that could help testing the hypothesis that the

amount/activity of fish liver PMAs may be influenced by waterborne exposure to a model

xenoestrogen — ethynylestradiol (EE2). We established the Bouin fixative as the best for

morphometric approaches, especially combined with the periodic-acid staining. Also, we

verified that the volume-weighted mean volume, the relative volume and the total volumes

of PMAs in the liver were reasonably fast stereological parameters that were to be used. A

simple image analysis approach for quantifying the amount of acid phosphatase in PMAs,

after histochemistry for detection, was used in parallel as a proxy on macrophage activity.

A first experiment was done with fish of each sex being held, separately, at 22ºC and

28ºC. A second followed where males were exposed to 75 ng/L of EE2, for 21 days. The

data show that sex matters, whatever temperature, when concerning some of the PMAs

parameters, which was indirectly supportive of the initial hypothesis. On other hand, and

contrarily, EE2 exposure did not exerted influences in the morphological parameters but

lowered the histochemistry signal. Overall, the study warns that the use of PMAs as

biomarkers of environmental contamination can be biased at least by sex related influences;

these should be well-known and controlled for a bioindicator fish species being considered in

experimental or environmental settings. Still, the study suggests that if fish liver PMAs are

modeled by xenoestrogens, with histological effects, then longer exposure periods would be

needed. Anyway, more studies are clearly needed for answering what is still at stake.

8

Resumo

Os agregados de macrófagos pigmentados (AMP) são grupos distintos de células

presentes no fígado, rim e baço de peixes, anfíbios e repteis. Foi demonstrado que estes

são modelados por factores endógenos bem como por estímulos exógenos tais como

poluentes. Por essa razão, alguns especialistas têm insistido que os AMP podem ser

usados como biomarcadores de stress ambiental, pelo menos de alguns poluentes pois

em certos casos podem não ser aptos devido à sua alta variabilidade e fraco

conhecimento da sua funcionalidade. Algumas publicações sugerem que, pelo menos em

peixe, os AMP podem ser influenciados por esteróides sexuais mas o assunto ainda

encontra pouco estudado. Em teoria, se tais efeitos modeladores se verificarem, tóxicos

que mimetizam os efeitos estrogénicos e/ou androgénicos podem actuar como

disruptores dos AMP. Para iniciar a abordagem de forma sistemática destas questões,

nós seleccionamos como modelo experimental um pequeno peixe com dimorfismo

sexual, o plátio. Como primeiro passo, nos quisemos estabelecer um método

estereológico imparcial para avaliar quantitativamente as alterações no tamanho e

conteúdo dos AMP no fígado. Usando este método, estudámos então os efeitos do

género do animal e de temperatura (temperatura óptima para procriação versus

temperatura não óptima) na quantidade e actividade basais dos AMP. Finalmente,

quisemos correlacionar estes dados com um primeiro ensaio que poderia ajudar a testar

a hipótese de que a quantidade e actividade dos AMP no fígado de peixe pode ser

influenciada por exposição aquática a um xenoestrogénio modelo – etinilestradiol (EE2).

Estabelecemos o Bouin como melhor fixador para analises morfométricas, em especial

quando combinado com a coloração de PAS. Verificamos também que o volume médio

ponderado pelo volume, o volume relativo e o volume total de AMP são parâmetros

estereológicos razoavelmente rápidos para ser aplicados. Um método simples de análise

de imagem para quantificar a quantidade de actividade da ácido fosfatase nos AMP foi,

após uso de histoquímica para detecção, usado em paralelo para ter uma aproximação

da actividade dos AMP. A primeira experiência foi realizada com peixes de ambos os

sexos, sendo mantidos separadamente a 22ºC e a 28ºC. Seguiu-se uma segunda

experiência onde peixes do género masculino foram expostos a 75 ng/L de EE2 por 21

dias. Os dados mostram que o género de animal é relevante, qualquer que seja a

temperatura, quando considerando alguns dos parâmetros dos AMP, o que é

indirectamente concordante com a hipótese inicial. Por outro lado, a exposição a EE2 não

exerceu influências nos parâmetros morfológicos mas diminui o sinal histoquímico obtido.

No geral, o nosso estudo alerta para a possibilidade da influência pelo género nos

9

resultados, quando se usa os AMP como biomarcadores; estas influências deverão ser

bem conhecidas e controladas se se quer usar os AMP de peixe como bioindicador em

testes experimentais ou em quadro ambiental. Ainda assim, o nosso estudo sugere que

para os AMP de peixe ser modelados por xenoestreogénios, com efeitos a nível

histológico, o tempo de exposição necessita de ser mais longo. Assim, mais estudos são

necessários para responder ao que ainda está em dúvida.

10

Introduction

Pigmented macrophage aggregates (PMAs), also known as melanomacrophage

aggregates or solely as macrophage aggregates, are distinctive groups of phagocytes,

typically loaded with a yellowish to brown/black pigment content, found in heterothermic

vertebrates, and are usually encountered within the reticulo-endothelilal supporting matrix

of hematopoietic tissues, namely spleen and kidney, and also in other organs such as the

liver and ovary (Agius and Roberts 2003; Manera et al. 2000). It has been suggested that

PMAs may be a primitive analogue of germinal centers of lymphatic nodes from mammals

and birds (Agius and Roberts 2003). They are usually nodular, with a delicate argyrophilic

capsule, and generally are close to vascular systems. At least in liver they do not seem to

differ in frequency between afferent and efferent blood vessels (Figueiredo-Fernandes et

al. 2007). The macrophages within the PMAs are normally tightly packed to form small to

large aggregates and their size increases after active phagocytosis of heterogeneous

materials, like those derived from hemolysis (Couillard and Hodson 1996; Agius and

Roberts 2003).

Despite of the appearance of PMAs at light microscopy level is well described in

literature, very little is known about their ultrastructure. It is known that they are very

complex with cells bearing indented nuclei and a large number of membrane bound

vacuoles, containing different types of material, mostly resulting from phagocytosis. Plus,

sometimes, in more developed PMAs, it can be seen the development of a capsule, made

of layers of flattened cells and noncellular material. This layer is a way to isolate the PMAs

from the surrounding environment (Agius and Roberts 2003). The distribution of PMAs on

the animal may vary with the species. Hartley and Treinies (1996) stated that in the most

phylogenetically recent bony fishes PMAs are more abundant in the kidney and spleen,

whereas in the more primitive fishes they are more present in liver. Despite this notion,

Thilakaratne et al. (2007), did not found PMAs on spottail shiners (Notropis hudsonius)

(Clinton, 1824) liver, they did found them in spleen.

PMAs are called so because of the presence of granular pigment. The contents

of the granular pigments varies, but the most frequent are melanin, hemosiderin and

lipofuscin (Agius and Roberts 2003; Bols et al. 2001), which can be correlated to the

phagocytic phenomena (Agius and Roberts 2003). The presence of melanin, one of the

most common pigments found in PMAs, is justified according to some theories as a

mechanism of self-defense against the free-radicals produced by the catabolism of fatty

acids (Agius and Roberts 2003) as well as the active sites of antibacterial processes by

11

producing hydrogen peroxide and related compounds (Agius and Roberts 2003). It was

initially thought that pigmented macrophages, unlike melanocytes, were unable to synthesize

melanin, but it was shown later that they can indeed do it (Agius and Roberts 2003).

Lipofuscin is the result of the oxidative polymerization of polyunsaturated fatty

acids, which can be a result of cell debris digestion or occur after dietary deficiencies; and

fishes are especially prone to this later subject (Agius and Roberts 2003). It is also called

the ―wear and tear pigment‖, as it accumulates with age, being one of the most common

pigments in PMAs of fishes (Agius and Roberts 2003).

As for hemosiderin, it is a brownish granular and insoluble pigment iron (ferric)

storing protein. It is related to hemoglobin catabolism from hemolysis and erythropoiesis

(Couillard and Hodson 1996; Agius and Roberts 2003), and therefore, high contents of

hemosiderin can be found after toxic exposure which induces hemolytic disorders after

hemolytic anemia (Couillard and Hodson 1996; Agius and Roberts 2003). Hemosiderin

granules are normally in close association with lipofuscin granules (Agius and Roberts

2003).

The distribution of these pigments within the several organs where PMAs are

present are not always uniform. Jordanova et al. (2008) found that, under natural

conditions, liver PMAs in Ohrid trout (Salmo letnica) (Karaman, 1924) females had more

hemosiderin and lipofuscin content and were most of the times devoid of melanin.

Furthermore, Kranz and Peters (1984) found that the majority of ruffe (Gymnocephalus

cernuus) (Linnaeus, 1758) spleen PMAs were filled with large amounts of hemosiderin,

while in liver PMAs fatty derivate inclusions were more commonly found. This suggested

that splenic PMAs are involved in the decomposition of effete blood cells while the liver

seems to undergo the role of storing toxic substances (Kranz and Peters 1984). Manera et

al. (2000) described in gilthead seabream (Sparus aurata)( Linnaeus, 1758) that the renal

PMAs are more abundant and much richer in eosinophilic content that the spleen PMAs.

More studies show that there are such differences in contents distribution among organs

and in fishes from the same taxonomic family (Leknes 2007).

As for their physiological regulation, PMAs seem to be affected by natural factors

like the breeding cycle (Jordanova et al. 2008) and age (Fishelson and Becker 2001).

PMAs were shown to maintain a fairly steady volume ratio in relation to the liver

parenchyma throughout all breeding cycle exception made for the post-spawning, where a

significant increase was recorded (Jordanova et al. 2008). Fishelson and Becker (2001)

studied the development of liver and pancreas in the domestic carp (Cyprinus carpio)(

Linnaeus, 1758) throughout a range of 15 years. They found that the aging process starts

12

to be measureable at about 1 year old, where the fat containing tissues begins to

increase. This increase, as well as worn-out and damaged cells triggers the immune

system namely the macrophage cell types, and hence the number and size of PMAs

increase (Fishelson and Becker 2001). Besides this, changes in PMAs proliferation are

somehow linked to feeding and living habits ( Montero et al 1999; Manera et al. 2000;

Mizuno et al. 2002; Rios et al. 2007), condition factor (Schwindt et al. 2006) and health

status of the fish (Agius and Roberts 2003). The presence or absence of food, and its

quality play a role at PMAs abundance. Starvation was studied on PMAs modulation on

wolf fish (Hoplias malabaricus)(Bloch, 1794) and masu salmon (Oncorhynchus masou)

(Brevoort, 1856). It was found that for continuous starvation, both number and area

increase as time passes by (Rios et al. 2007; Mizuno et al. 2002). Other effects of diet like

the effects of iron, zinc and copper enriched diets were also analyzed (Manera et al.

2000). It was also shown in ruffe a correlation between tumorous nodules and the number

and size of PMAs in spleen (Kranz and Peters 1984). So, different patterns of PMAs may

be found in fish according to inherent natural conditions and respective effects.

Environmental inputs can also model PMAs proliferation. Parasites, like protozoa

and bacteria can also induce an increment in PMAs numbers (Couillard and Hodson,

1996; Thilakaratne et al. 2007). More, many studies have shown the effect of exogenous

compounds on PMAs proliferation on fish. The size of PMAs was positively correlated with

polychlorinated biphenyl (PCB) exposure in carp as well with the cytochrome P450 1A2

activity; however, the number of PMAs was not statistically different (Fisher et al. 2008). In

the same line, effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on PMAs proliferation

were observed on juvenile carp. TCDD increases the number of PMAs in spleen as well

as reduces the level of hemoglobin, adding to the hypothesis that hemolysis is a

contributing factor for the PMAs regulation (van der Weiden et al. 1994). Chromium

exposure on plaice (Pleuronectes platessa) (Linnaeus, 1758) makes the number of spleen

PMAs to triple in number while reducing their mean area (Kranz and Gercken 1987).

Environmental mixtures were also used to understand how PMAs behave to

environmental stress. Influences of paper mill effluents have been also studied. Couillard

and Hodson (1996) conducted an epidemiological study on fish living downstream a paper

kraft mill. High levels of lipofuscin were found in fish living closer to the mill, in parallel with

an increase of the PMAs density. The high lipofuscin levels found combine with cyp5A

data retrieved from the epidemiological study, and the known hemolytic effects of resin

acids, lead to the hypothesis that the increment on PMAs density was due to hemolytic

and lipid peroxidation phenomena (Figure 1) (Couillard and Hodson 1996). On the other

hand, Van den Heuvel et al.(2005) performed a laboratory study using juvenile rainbow

13

trout (Oncorhynchus mykiss) (Walbaum, 1792), and it was found a significant increasing

dose-effect relation on PMAs density in spleen (Van den Heuvel et al. 2005).

Chronic crude oil exposure was found to increase PMAs in spleen and kidney on

Atlantic codfish (Gadus morhua) (Linnaeus, 1758) (Khan and Kiceniuk 1984). Sewage

sludge effects were also measured. It was discovered an increment in number of PMAs in

both spleen and kidney in dab (Limanda limanda) (Linnaeus, 1758) (Secombes 1991).

Field studies also support the hypothesis that pollutants have effects on PMAs regulation.

Along the St Lawrence River, Canada, spottail shiners (Notropis hudsonius) (Clinton,

1824) were studied and had a battery of biomarkers measured where PMAs were affected

by the pollution, having higher counts in the more polluted areas (Thilakaratne et al.

2007). In the USA, a relation between mercury bioaccumulation and PMAs proliferation

was also found (Schwindt et al. 2008).

Fish are used as monitoring species in ecotoxicology, as they are able to provide

valuable data about their environmental conditions. PMAs may also be useful as non-

specific biomarkers, as several studies suggest (Schwindt et al. 2006). Some authors

conclude that changes PMAs after pollutant exposure are a good biomarker, together with

other morphological indicators (Fishelson and Becker 2001; Manera et al. 2000). But while

some authors are strongly optimistic about their suitability as biomarkers, others are not

so. As said before, several natural factors model the PMAs proliferation, as seasonal

fluctuations (Jordanova et al. 2008), and they are non-specific, which can make it difficult

Figure 1 Diagram demonstrating the hemolytic and oxidative damage that Craft mill effluents has and how they affect the PMAs density on fish.

Exposure to

Craft Mill

effluent

Hemolysis

Hemossiderin

Increased density

of PMAs

Induction of cyp1A

and production of

superoxide ions

Lipo-peroxidation

of cell membranes

Lipofuscin

14

to judge the retrieved data (Montero et al. 1999). Haaparanta et al. (1996) warned for four

main faults on PMAs suitability as biomarkers. First, there is considerable variation in

PMAs among individuals in the same species. Second, different species reveal different

natural patterns, which makes it difficult to establish base values for a wide number of

species. Third, in the same animal, differences can be spotted comparing different organs.

Not just differences on number or size, but as well on pigment content. Moreover, these

differences can reveal seasonal patterns. Fourth, the relationship between PMAs and

environmental pollution is far from clear. How much they induce proliferation or how they

do it are subjects who still need further study. Also, as expected, PMAs number should

increase with exposure, but we can also expect reduction in size or in number, if those

pollutants decrease immunocompetence (Haaparanta et al. 1996).

In this study, we start to address the question if fish liver melanomacrophages

may be modulated by xenoestrogenic and xenoandrogenic pollutants. For this aim, we

initially sought to look for the influence of baseline physiological aspects that could offer

clues for an endogenous regulation of PMAs by sex steroids. Firstly, we manipulated the

water temperature so to either cause or not gonadal ripening in both sexes, and therefore

induce or not endogenous production of sex steroids. Secondly, we focused on the effects

on PMAs of an endocrine disruptor compound (EDC), a synthetic estrogenic hormone, the

ethynylestradiol (EE2). An EDC is ―an exogenous substance or mixture that alters

function(s) of the endocrine system and consequently causes adverse effects in an intact

organism, or its progeny, or subpopulations‖ (Mills and Chichester 2005). These

compounds mimic the endogenous endocrine compounds, interaction with its receptors

and changing the normal function of the endocrine system. Other mechanisms on

biomolecular level have also being discovered (Tabb and Blumberg 2006). It was shown

that EDC exposure in guppies lead to a decrease in population reproducibility (Mills and

Chichester 2005). Besides the potential environmental meaning of such alterations, from

an economic viewpoint valuable fish for mankind exposed to EDCs may have their stocks

depleted much faster than it would if they were not exposed (Mills and Chichester 2005).

Among these, we may name the disruption on the natural hormone metabolism, protein

degradation, sensitization, methylation of DNA, effects on receptors other than estrogen

receptors and androgen receptor, and others. EE2 is a known EDC and it binds with

estrogen receptors in exposed organisms with an affinity identical or greater to the

endogenous estrogen hormone, 17β-estradiol, and have the potential to exert estrogenic

effects at extremely low concentrations (Mills and Chichester 2005). EE2, as well as other

EDC, end up in the environment from human activity. EE2 used on contraception pills is

15

excreted by humans and animals and thus enter the environment through the discharge of

domestic sewage effluents and disposal of animal waste (Yin et al. 2002).

The reproductive physiology of vertebrates in both mammalian and non-

mammalian is fairly similar. All the broad structure and function of the reproductive axis

involving the hypothalamus, pituitary and gonads are conserved (Figure 2). In all

vertebrates, the release of a decapeptide gonadotrophin-releasing hormone from the

hypothalamus stimulates the pituitary to secrete gonadotrophic hormones. These signal

the gonads to synthesize steroid hormones.

The basic biosynthetic pathways for steroid hormones and the active steroid

hormones themselves are also well-conserved in vertebrates (Arcand-Hoy and Benson

1998). However, fish have some aspects of reproductive physiology that may cause them

to respond differently to EDCs than mammals. For example, in fish, secretion of GnRH

normally changes in response to environmental clues, whereas in mammals do not

(Dawson 1998). This fact is well illustrated by the readiness in fish from the poeciliidae

family for reproduction as determined by optimal environmental temperature (Vondracek,

Wurtsbaugh, and Cech 1988). In poeciliidae, at the right temperatures, a higher amount of

energy is driven to reproduction purposes, such as gonad development and offspring

generation. In contrast, at lower temperatures, energy supply is preferentially allocated for

survival and growth purposes (Vondracek, Wurtsbaugh, and Cech 1988). It is then

Figure 2 Diagram about the sexual regulation on fish. Any change on any of the pathways will usually result on a increase or decrease on the sexual related developments. GhT: gonadotropins (GhT I = Teleost FSH; GhT II = Teleost LH).

Hypothalamus

Pituitary

Synthesis of

sex steroids

Maturation of

gametogenesis

Gametogenesis

Steroidogenesis

GhT I GhT II

Phenotype

behavior

16

expected that at higher temperatures greater amounts of natural sexual hormones are

present in the animal, giving us a way to tune the estrogenic and androgenic levels on the

fish, by just changing the environment temperature and keep the same energy input.

Herein we performed the experiments using the southern platyfish (Xiphophorus

maculatus) (Günther, 1866) for several reasons. First, they are easy and cheap to keep in

large numbers in normal laboratory conditions. They are very resilient to environment

changes, which proved useful for testing different temperatures and xenobiotics. Platyfish

have being used for long time in genetic and other biological investigations. Despite this,

there is a lack of knowledge in these the matters we want to address in this particular fish.

Based on the fact that most PMAs contain melanin, lipofuscin and hemosiderin,

we searched for staining techniques which would target these particular pigments and

make them possible for optical microscopy analysis. Melanin has high affinity to silver

nitrate, and hence, the Masson-Fontana staining method was taken into consideration

(Bancroft and Stevens 1990a). As for lipofuscin, hematoxylin and eosin is the most used

technique. But, as lipofuscin mesh contains sugars, among other things, the periodic acid

Schiff reaction (PAS) also presented it viable for testing (Bancroft and Stevens 1990b).

Finally, hemosiderin is best put into evidence with the Perl’s staining protocol (Bancroft

and Stevens 1990c).

Leukocytes and macrophages have enzymes to help them perform their role.

Each cell type has on its own some specific set of enzymes. The most common targeted

enzymes used to identify the cell type are addressed in Table 1 (Lin et al. 2005).

Regarding the macrophages, it is possible to see that among all the enzymes tackled only

acid phosphatase (AP) is present and stains positive with proper techniques (Lin et al.

2005).

Table 1 Demonstrative chart of which enzymes are present in each type of cells of the immune system. AP: Acid Phosphatase; NBE: α-Naphthyl Butyrate Esterase; BG: β-Glucuronidase.

Cell Type

Enzymes present

AP NBE BG

Monocytes + + -

Macrophages + - -

Neutrophils + - -

T Cells + + +

B Cells + - +

17

In conclusion, spleen and kidney PMAs has been often studied in fish, but when

it comes to liver, fewer studies were made and most data must be extrapolated from other

organs to liver. However, this organ has complete different and unique functionalities. So

any study offering insights on the regulation and content of liver PMAs is valuable to fully

understand the PMAs role in that organ. Additionally, the changes in the contents of PMAs

vary in response to several stimuli (Bols et al. 2001). As stated above, some of the

contents of PMAs result from recycling processes like erythropoiesis, and if

environmentally stimulated it is expected that hemosiderin and lipofuscin levels increase.

Hence the study of PMAs may give us insight about both the physiological status and the

environmental conditions where fish live. The latter may be challenging due to

environmental stressors, including exposure to EDCs. Toxicological impacts on PMAs in

fish, along with the role of natural androgens and estrogens on PMAs, are poorly studied.

So, we aim to disclose new data that may help understanding how PMAs are regulated by

sexual hormones, and therefore by the sexual status of the animal, while investigating

eventual disrupting effects of environmental contaminants that may mimic the

endogenous estrogens or androgens. In detail, and using the platyfish as model, we

aimed in this first approach to: 1) establish and implement an unbiased quantitative

method to evaluate histopathological changes in the amount of fish liver PMAs for use

both in experimental assays and in biomonitoring; 2) study the effect of sex and

temperature (breeding optimal versus lower non-optimal) on the baseline liver content of

PMAs; and 3) test the hypothesis that the amount/activity of fish liver PMAs may be

influenced by waterborne exposure to a model xenoestrogen.

18

Material and methods

Establishment of better fixative solution

Two fishes were sacrificed by severing the spinal cord after an overdose of

anesthetic (phenoxyethanol (Sigma™)). Then, their livers were collected and measured.

Their livers were then split in two, totaling 4 liver pieces. Each piece was then placed for

12h in a different fixative: 10% neutral buffered formalin (JT Baker™); 4%

paraformaldehyde, in 0.1M phosphate buffer (pH 7.4, Merck™); Bouin’s solution

(Panreac™); and Davidson’s solution, lab made.

The sampled pieces were routinely dehydrated through an increasing gradient of

alcohols, cleared in xylene, infiltrated, and finally embedded into paraffin blocks. These

were cut in 5µm thick sections, using an automatic microtome Leica 2155, and then

stained with H&E. The tissues were then observed regarding their correct preservation

and suitability for image analysis, namely of the PMAs.

Establishment of the best staining technique for image analysis

Four fishes were sacrificed by severing the spinal cord after an overdose of the cited

anesthetic. They had their livers removed and preserved in 10% neutral buffered formalin

for 12h. The sampled pieces were routinely dehydrated through an increasing gradient of

alcohols, cleared in xylene, infiltrated, and finally embedded in paraffin. Liver paraffin wax

blocks were cut in 5µm thick sections in an automatic microtome Leica 2155. Each liver

was then stained by one of the following techniques: H&E, Masson-Fontana, Perl’s

Prussian Blue and PAS.

Deparaffinization and mounting of sections

All liver sections went through the same process before being stained. Liver

sections were deparaffinized in xylene and then re-hydrated in a decreasing gradient of

alcohols and rinsed in tap water and finally also in distilled water. After this point, sections

19

were ready to be stained. After staining, the same method was also applied to all slides

which were mounted. After being cleaned in tap water, sections were dehydrated in an

increasing gradient of alcohol and cleared in xylene. Slides were then mounted using DPX

mounting medium. The following protocols were applied in the middle of these two steps.

Hematoxylin and eosin

After deparaffinization, liver sections were immerse in hematoxylin solution for 4

minutes and washed in tap water. Again, sections were immerse in eosin for one minute,

and then mounted.

Masson-Fontana

After deparaffinization, slides were kept in ammoniac silver nitrate for 4h, protected

from the light. After that, slides were washed with distillated water and stained with light

green for a minute. They were washed again in tap water and dehydrated. The slides

were then mounted using DPX mounting medium.

Perl’s prussian blue

Liver sections were deparaffinated. Meanwhile, Butting solution was prepared by

mixing equal parts of chloridric acid 2% and potassium ironcianate 2%. Once this solution

was made and warm up to 60ºC, slides were immersed and kept like this for 10 minutes at

60ºC. After, they were washed in distilled water and stained with eosin for contrasting.

They were washed again in tap water and dehydrated and then mounted using DPX

mounting medium.

20

Periodic acid Schiff reaction

Liver sections were dewaxed to distillated water. Liver sections were then oxidized

with periodic acid 0.5% for 7 minutes. They were washed in tap water and in distillated

water. Schiff reagent (Sigma™) was then applied to slides for 10 minutes and slides were

washed till waster presented clear and colorless. Slides were then immersed into

hematoxylin for contrast, for 4 minutes. They were washed again in tap water and

dehydrated in an increasing gradient of alcohol and cleared in xylene. Slides were then

mounted using DPX mounting medium.

Establishment of baseline sexual and gonadal maturation influences

From a batch of 80 previously acclimated fishes, forty fishes, twenty males and

twenty females, were selected and kept in separated 4 tanks, ten units per tank. Each

tank had only one sex, either male or female, totaling 2 male tanks and 2 female tanks.

These fishes were all kept under same conditions of photoperiod (12 h/12 h), temperature

(26ºC), and were feed to satiation (TetraMin, Tetra™). The animal density was kept equal

as well what concerns ventilation and filtration. After this period of fifteen days of

acclimatization to the new conditions, temperature in two of the four tanks (one with males

and one with females), was reduced to 22ºC, while in the other two remaining tanks it was

increased to 28ºC. All the other conditions were kept. Temperature was

decreased/increased in 1ºC a day. Fish stayed in these conditions for 15 days, with water

quality being monitored each other day, for nitrites, ammonium and pH. Temperatures

were measured daily.

Sub-chronic exposure to a xenoestrogenic compound

From a batch of 30 previously acclimated male fish, 2 tanks were filled with 15 fish

each. These were kept for 15 days under equal conditions of photoperiod (12 h/12 h),

temperature (26ºC), and feeding to satiation (TetraMin, Tetra™). Animal density was also

kept identical as well as air pumping and filtration. After this period of acclimatization,

temperature was reduced to 22ºC (1ºC daily). For the exposure experiment purpose, the

first tank was used as solvent control, and the second one was kept with EE2 at a nominal

21

concentration of 75 ng/L. Fish were exposed to these conditions for 21 days, in a

semistatic regime. Water and EE2 concentration were renewed daily. At same time, water

quality was monitoring by analyzing the pH, ammonium and nitrite levels every each

week. Temperature was measured and recorded in a daily basis.

Tissue sampling, processing, and analyses

Fishes were firstly sacrificed by severing the spinal cord after an overdose of

anesthetic (phenoxyethanol (Sigma™)), and then their body weight and lengths were

measured. Right after, dissection was made with liver and gonads being sampled and

weighted. The liver was then cut in two halves and one was weighted and reserved to light

microscopy, while the other was set aside for enzymatic histochemistry assays. The

collected pieces of liver and gonads for light microscopy were then kept in Bouin fixative

for 24h, which was then replaced by ethanol 70%, while the collected pieces for enzymatic

histochemistry assays targeting AP activity were embedded in OCT medium (Leica ™),

then frozen in liquid nitrogen, and finally kept at -70ºC.

For light microscopy analysis, the sampled pieces were routinely dehydrated

through an increasing gradient of alcohols, cleared in xylene, infiltrated, and finally

embedded into paraffin. Liver and gonad paraffin wax blocks were serially cut in 5 µm

thick sections, using a gapping of 20 µm or 10 µm between the sections selected for

staining, according to sample size. Some sections were stained with H&E for general

visualization whereas the majority was stained by PAS and counterstained with

hematoxylin for PMAs analysis. With this staining, PMAs stains in deep pink, while the

rest of parenchyma remains bluish.

Paraffin sections of liver and gonads were used for qualitative and quantitative

analysis. In the liver, the identification was based on the characteristics of the PMAs at

histological level, namely, the development degree (Manera et al. 2000). By the use of

stereology, the relative (VV) and total volumes (V) of the PMAs in the liver were estimated.

22

Enzymatic histochemistry assays

For enzymatic histochemistry assays, the frozen samples of liver were sectioned

in a cryostat (CM1850 Leica™), first, with a 5, 8 and 10 µm thickness for testing which

thickness was best, and then latter cut only at 8 µm. Cabin temperature was -25ºC.

Meanwhile, four solutions were prepared to make the incubation solution. The first

solution was the substrate solution (a). The substrate was naphtol AS-BI phosphate

(Sigma™) 1% (m/m) in Dimethyl formamide (Sigma™). Then the buffer solution was also

made, by dissolving 1.17 g of anhydrous sodium acetate in 2.9% (m/m) barbital aqueous

solution (b). A 4% (m/m) sodium nitrite solution (c) was also prepared. Finally, the staining

solution (d) was made by dissolving 1 g of pararosanilin (Sigma™) in 20 mL of water

previously acidified with 5 mL of hydrochloric acid (conc.). The final incubation solution

was made by mixing 0.5 mL of (a), 2.5 mL of (b), 0.4 mL of (c) and 0.4 mL (d), previously

mixed in equal parts. Finally, 6 mL of water was added to get a final volume of

approximately 10 mL. The tissue sections were incubated in the previously prepared

solution. They were then washed in tap water, dehydrated, and mounted with DPX

mounting medium. The first sections were incubated for 5, 10 and 15 minutes for

determination of the best staining quality. From then on, we used 5 minutes.

Stereological and histoenzymatic analysis

From each liver block, 10 to 15 slides were selected. In each, systematically

sampled fields were quantified using an inverted light microscope Olympus IX71 equipped

with a motorized Prior ProScan II frame and an Olympus Camera DP71. These elements

were coupled with an image analysis (VIS - Visiopharm Integrator system V. 3.6.0.0,

including the newCAST – new Computer Assisted Stereological Toolbox). The fields were

spaced 400 µm and 400 µm, respectively on the X and Y axis, enough for sampling about

23% of the total section. Standard stereological techniques based on manual point

counting (Hfreere and Weibel 1967) were used to estimate the relative volume of all the

PMAs within the organ, where the ratio of points targeting PMAs to points for all liver was

1 to 32. The relative volume was then calculated according to the following formula:

23

For the liver volume estimation, the Cavalieri method was used (on the sampled

half of the organ). Sequentially, liver sections were systematically sampled and manual

point counting was performed to estimate each section area. Then, as the gap between

sections was known, the total volume was estimated according to the subsequent formula:

The ―2‖ on the formula represents an approximation to the ratio of analyzed liver

to total liver. As liver were split in halves, the ratio is about 0.5 and hence we have to

double to obtain the total liver volume. Once the liver volume has been estimated, the

PMAs volume could be estimated too, according to the following formula:

For histoenzymatic analysis, images were evaluated for their color intensity. The

examination was measured the by measuring the color saturation in image software

(Adobe Photoshop CS 4) on channel R 204; B 0; G 0. Only High Temperature Females

and Low Temperature Males, i.e., the two expected extremes in endogenous estrogenic

content were analyzed.

Individual PMA volume-weighted volume measurement

Using the image analysis toolbox and software CastGrid (Ver. 2.0, Olympus), at a

final magnification of 799x, the images were scanned for sampling PMAs. In each field, a

grid with parallel lines having sampling points (defined by crossed lines) was overplayed.

Each time the crossed line intersected a PMA, the length of the intercept within the PMA

boundaries was measured (Figure 3). As the PMAs are roundish and also do not display

any preferred orientation in 3D within the liver, the intercept segment is viable. That

length, l, was then used for estimating the so-called volume-weighted volume of the PMA,

using the following formula:

24

Figure 3 Example on how the volume-weighted volume was calculated. The dark lines with the small grey slashes make the sample grid overlay. Whenever a PMA touches the grey slash, the length of the dark line (the intercept) is measured (pink on figure) and l is hence derived to compute the volume. Liver section stained with PAS.

Immunohistochemistry staining for vitellogenin

To find out whether or not EE2 had exerted a baseline physiological effect in the

fish, we performed an immunohistochemistry assay for vitellogenin in hepatocytes (Vtg).

An indirect, two-step method was applied. A pair of slides, one from each of the

experimental groups, plus one slide for positive control were dewaxed in xylene and then

re-hydrated in a decreasing gradient of alcohols and rinsed in tap water and finally in

distilled water. Antigen recovery was needed and performed with a microwave oven (Y53

Moulinex™), using a bath of a metal rich solution. The slides were then rinsed in tap water

and in distilled water, followed by blockage of endogenous peroxidase via contact with a

blocking solution (Merck™). The slides were once again washed in water and in

phosphate buffer solution (PBS, Laboratory made). To minimize non-specific staining

sections were immersed in a blocking reagent (Invitrogen™).

With the aid of a hydrophobic pen, contingency zones were marked around the

sections where the primary antibody was applied, with a dilution of 1/1000. Incubation

time was 12h and done in a wet chamber, at room temperature (~22°C). After incubation

25

the slides were washed again in PBS three times and the second antibody (Invitrogen™)

was applied, being incubated for 15 min at room temperature in a wet chamber. After

being washed in PBS, sections were covered with streptavidin-peroxidase complex

(Invitrogen™), for 15 min, in a wet chamber at room temperature. Meanwhile a DAB

solution (DAB Substrate Buffer & Liquid DAB Chromogen – DAB057) was freshly

prepared and applied to the sections for 1 to 5 min, having the slides under monitoring.

After the above, the slides were washed in tap water, stained with hematoxylin

for 1 min, washed again in tap water and de-hydrated in an increasing gradient of alcohol,

and cleared in xylene. Slides were then mounted using DPX medium.

Statistical analysis

For the statistical analysis, the IBM SPSS Statistics19 software was used. In the

first assay, descriptive analyses were made on data collected using the different

temperatures and sexes as factors. Normality was checked as it is required to perform

analysis of variance (ANOVA). If the data did not follow a normal distribution, adequate

transformations were made to achieve normality; namely base 10 logarithmic

transformations. ANOVA were performed with a level of confidence of 95%. Post-Hoc

tests were also made, namely those of Tukey and Newman-Keuls. Whenever one of the

tests disclosed a significant difference and the other did not, we faced the result as

marginally but nevertheless significant. This strategy better helped to biologically interpret

the data.

For the second assay, descriptive data analyses were made using the exposure

condition as factor. Normality and homogeneity of variances was made. Whenever it was

necessary for a parameter, base 10 logarithmic transformations were applied to data sets.

As there were only two test groups, statistical analyses were made using the independent-

sample Student's t-test, with a confidence level of 95%.

26

Results and discussion

Establishment of best staining and fixative solutions

As PMAs are known to have melanin, hemosiderin and lipofuscin, staining

techniques targeting these pigments were tested to found which one was more suitable

for the platy liver. The basic H&E offered only a very gentle contrast of PMAs against the

rest of the parenchyma (Figure 4a, 4b), which means that it would be an inadequate stain

for image analysis, as this typically requires a good contrast on the targeted elements.

Figure 4 a: Male liver stained with H&E. Two PMAs are visible (thicker arrows), next to blood vessels (star), Lipofuscin pigment is visible even at low magnification (thin arrow) b: Male liver section stained with H&E. Two PMAs (thicker arrows) presenting well defined lipofuscin pigments (thin arrows) are spotted next to blood vessels (star). Images c and d: Male liver sections stained with Masson-Fontana. A small PMA is visible at the thicker arrow. Thin arrow indicates small melanin pigments. Star: Blood vessel.

The Masson-Fontana staining was used to stain melanin, and although it properly

stained melanin, very few PMAs had melanin, thus making it quite difficult to spot and

then quantify PMAs that would not stain. Also, the counterstaining effect, with light green,

27

gave little contrast against the PMAs, especially those without melanin (Figure 4c, 4d).

The same scenario was found with the Perl’s staining, which aims highlighting the

hemosiderin. Only very few PMAs presented this pigment and stained positive (Figure 5a,

5b). For this reason we had to discard this staining method for additional analyses.

Figure 5 a: Liver section stained with Perl’s prussian blue. One PMAs are visible (thicker arrows) next to vessels (star) Hemosiderin pigment is visible stained blue (thin arrow). b: Liver section section stained with Pears. PMAs visible at thicker arrow. Hemosiderin faintly stained at thinner arrow. c: Liver section stained with PAS. A PMAs is visible at thicker arrow. Thin arrow indicates several lipofuscin pigments stained. Star: broken tissue where vessel was. d: Large PMAs stained with PAS where are visible, due to fragmentation, several macrophages and they lipofuscin content (thin arrows). Star: space due to tissue shrinkage and blood vessel.

The last staining we tested aimed to reveal sugars known to be present among the

lipofuscin mesh (Monserrat et al. 1995). The PAS reaction, counterstained with hematoxylin,

was the staining technique that proved both reliable, as stained all the PMAs, and it was

easy to spot in the microscope, as PMAs were well contrasted against the parenchyma

(Figure 5c, 5d). Moreover, this method proved to be one of the simplest to apply. For all

these reasons, the PAS was chosen for all the subsequent image analyses.

Four fixative solutions were tested, briefly: 10% formalin, 4% paraformaldehyde,

Bouin and Davidson. The worst preservation observed was that found on the liver fixed

with 10% formalin. With this fixative, the outer regions of the liver were prone to break

28

apart, especially when mounting the slide (Figure 6). The Davidson’s solution was also

inadequate, as it caused widespread breaks in the sections (Figure 7). The second best

preservative was the paraformaldehyde. Although offering good preservation of the

tissues, the PMAs shown some dismemberment, and the macrophages dissociated from

each other (Figure 8). The Bouin’s solution was the fixative of choice, as it undoubtedly

provided the best results, with virtually no artifactual breaks in tissues and with the PMAs

looking intact (Figure 9).

Figure 7 Liver fixed with Davidson’s solution. a: The shrinkage effect is pronounced and visible in all the section. PMAs indicated by arrows, despite hardly seen at low magnification. H&E. b: A PMA is better visible, intercepted by an artifactual break (arrow). H&E

Figure 6 Liver fixed with 10% formalin. a: PMAs are shown with arrows at very low magnification. Star shows a zone of broken tissue. H&E. b: Zooming of the area displayed in a, where it can be seen the relatively poor preservation. A PMA with melanin is pointed by an arrow. H&E.

29

Given the results, the Bouin’s fixative was chosen for the subsequent histological

analyses, as it preserved both parenchyma and stromal elements, including the PMAs,

consistently offering a good aesthetic appearance, and making the image analysis easier

and more reliable, as identification was flawless and no artifacts/distortions were present.

Establishment of baseline sexual and gonadal maturation influences

During the trial, only one casualty was reported in the high temperature males. The

mean temperatures recorded were as followed: High temperature females 28.1 ± 0.8ºC;

Low temperature females 22.8 ± 1.0ºC; High temperature males 30.2 ± 0.7ºC; Low

temperature males 22.5 ± 0.9ºC.

The body morphometric data retrieved at sampling are shown in Tables 3 and 4.

From the baseline data we calculated the hepatosomatic index (HSI) and the gonodosomatic

Figure 9 Liver fixed with Bouin’s solution. a: Preservation is consistently of good quality. Arrows point to some PMAs, discernible at low magnification. H&E. b: Zoomed in PMA. PMAs shows to be intact (arrow) H&E

Figure 8 a: liver fixed with 4% paraformaldehyde. An overall good preservation was attained. PMAs can be spotted in and outside the liver (arrows). b: Zoomed in PMA showing some degradation on PMA structure. H&E

30

index (GSI). We had at low temperature males a HSI of 2.17 ± 0.40% (mean ± SD) and a

GSI of 1.30 ± 0.37%, at low temperature females we had a HSI of 2.51 ± 0.87% and a

GSI of 5.63 ± 4.15%, at high temperature males we had a HSI of 1.88 ± 0.31% and a GSI

of 1.13 ± 0.63%, and, finally, at high temperature females we had a HSI of 2.54 ± 0.66%

and a GSI of 4.34 ± 4.30%. In a sex, no significant differences existed for both indexes.

At each temperature, animals of the same sex did not show statistically significant

differences in relation to their total lengths or weights (Post-Hoc: Tukey, p>0.05). On the

other hand, differences were found in the gonad weight between sexes (post-Hoc: Tukey;

p<0.05) (Figure 10). This was expected as fish females usually have larger gonads (at

least when adults), and additionally some of the females were pregnant as seen in Table

2. This fact led to a high variation on the ovary weights, as the fish were not synchronous,

i.e., they had different pregnancy stages at sampling time. The males presented less

dissimilarity, being their gonads much more homogeneous in size. As for the GSI, it showed

differences between males and females sexes (Post-Hoc: Tukey; p<0.05), which followed

the same logic as the gonad weight. Our data show that despite fish were kept separated

by sexes for a relatively long period before sampling (about 1 month), the fact is that some

females were still able to use stored spermatophores for later fertilization and develop

pregnancy. This was not really unexpected, as females seem to be able to store viable

sperm for up to several months within the ovary and gonoduct (Potter and Kramer 2000).

From an operational point of view, and if any confounding effects of the pregnancy status

are to be removed, longer acclimation with isolated sexes may be equated in the future.

Table 2 Pregnancy stages on females sampled: (-) Not pregnant; (+) Early pregnancy; (++) Middle pregnancy; (+++) Late pregnancy.

Fish Low Temperature Female High Temperature Female

1 (-) (-)

2 (++) (++)

3 (+++) (+++)

4 (+) (-)

5 (-) (-)

6 (++) (+)

7 (++) (-)

8 (+) (+)

9 (-) (++)

10 (+) (-)

31

Table 3 Body and organ related gross measurements of fish acclimated to low temperature

Length (cm) Body Liver Gonad

Standard Total Total Weight (g) Total Weight (g) Weight for MO (g) Remaining Weight (g) Total Weight (g)

Low Temperature Males

Mean 3.54 4.26 1.450 0.032 0.017 0.015 0.018 Standard Deviation 0.28 0.30 0.441 0.013 0.007 0.007 0.005

Variation Coefficient 0.08 0.07 0.304 0.406 0.412 0.467 0.278

Low Temperature Females



Table 4 Body and organ related gross measurements of fish acclimated to high temperature


Standard Total Total Weight (g) Total Weight (g) Weight for MO (g) Remaining Weight (g) Total Weight (g)

High Temperature Males



High Temperature Female



32

Figure 10 Graphic chart of gonad weight vs. experimental group. Two different statistical settings were found (a and b; p<0.05). On set a we can find males together with the high temperature female, while in set b we can only find females. Box plot graphic with minimum, first quartile, median, third quartile and maximum. Open circles represent statistical outliers (herein not excluded from the analysis).

In relation to the liver weight we also found significant statistical differences. Like in

gonad weight comparisons, differences were found between males and females, being

the values higher in females (Post-Hoc: Tukey; p<0.05) (Figure 11), and namely in those

held at lower temperatures. The bigger livers seen in females may be associated to the

needs for a higher metabolism, namely connected with breeding. While males were on a

status of low stress and had low demands on metabolism, the same cannot be said about

females. Most of them were pregnant, which logically means an increase in metabolic

activity, producing new ―raw materials‖ for incorporation in new offspring. This somehow

indicates us that liver grows in parallel to the embryos in the ovaries of pregnant females

(Vondracek, Wurtsbaugh, and Cech 1988). This may be due to the fact that in Poeciliidae

fish, like platyfish, the embryos do not rely solely on the egg yolk to grow, as there is

transition of material from the mother to the offspring, to help them grow (Marsh-Matthews

et al. 2009). In order to process this excess of metabolites, the liver undergoes some

hepatomegaly phenomena. As for the HSI, the statistical analyses reveals that HSI shows

33

no differences between different groups, unlike liver weight (Post-Hoc: Tukey, p>0.05).

This corresponds to what was said above, i.e. the livers follow up the gonad development

in a proportional way. As for the tendency to have bigger livers on lower temperatures,

there was indeed a statistically significant difference between both temperatures, but only

if sexes were grouped (t-test p<0.05). At this time we can only speculate about the

underlying reasons. This data follows somehow the data described by Vondracek et al.

(1988). In higher temperatures the energy surplus is driven for reproduction purposes

such as gonad ripening, offspring and mating. Therefore, higher amount of energy is used

for these purposes, leaving less for growth and less to be stored as adipose tissue or as

lipid reserves in the hepatocytes. Consequently, the fish livers at lower temperatures

could have more volume due to less energy being spent on reproduction. Also, these

results may be due to activation of some biochemical pathways only activated under such

temperatures, leading to an increase and decreased of liver metabolism and liver volume,

either by increasing the number of cells or the volume of these, or even both. In any case,

further study needs to be addressed to this mater to fully understand the real reasons of

such variation and the underlying cellular kinetics. Finally, it is informative (revealing of a

consistent sampling procedure) to mention that the weight of the liver pieces processed

for light microscopy had no statistically significant differences (Post-Hoc: Tukey; p>0.05).

34

Figure 11 Graphic chart of liver weight vs. experimental group. Two different statistical settings were found (a and b; p<0.05). On set a we can find males together with the high temperature female, while in set b we can only find females. Box plot graphic with minimum, first quartile, median, third quartile and maximum. Open circles represent statistical outliers (herein not excluded from the analysis).

The data from the image analysis regarding volume measurements can be found in

Table 5. No statistical difference was found in the liver volumes (Post-Hoc: Tukey,

p>0.05). This follows the analysis made on the weights of the liver pieces collected for

stereology. In contrast, differences were found in both relative and absolute volumes of

PMAs.

As to the relative volumes of PMAs we found that females had higher percentages

than males. Only high temperature males could be statistically grouped together with

females (Figure 12). Once again, females showed more variability, eventually due to the

asynchrony of ovary maturation and pregnancy status, and consequently of the sex

hormones profile.

35

Figure 12 Graphic chart of relative PMA volume vs. experimental group. Two different statistical settings were found (a and b; p<0.05). On set a we can find females together with the high temperature males, while in set b we can only find females. Box plot graphic with minimum, first quartile, median, third quartile and maximum. Open circles represent statistical outliers (herein not excluded from the analysis).

36

Table 5 Liver volume, and relative and total volumes of PMAs of fish acclimated to low and high temperatures.

Low Temperature Males

Low Temperature Females

High Temperature

Males

High Temperature

Females

Liver Volume (mm3)

Mean 10.669 12.515 10.770 13.293

Standard Deviation 4.883 7.113 4.345 4.523

Variation Coefficient 0.458 0.568 0.403 0.340

Relative PMA Volume (VV)

Mean 0.16% 0.47% 0.42% 0.69% Standard Deviation 0.11% 0.37% 0.35% 0.48%


Absolute PMA Volume (mm3)

Mean 0.12 0.06 0.05 0.09

Standard Deviation 0.19 0.10 0.04 0.08


Using the liver volume and the relative PMA volume, we estimated the absolute

PMA volumes (Figure 13). For the latter we found three statistical sets in the SPSS

output. On one set we find the females, on the other the males, and in the third one, we

find high temperature males and low temperature females. These results indicate us that

temperature does have an effect on the amount of PMAs and, indirectly, agrees with the

possibility of sex steroid regulation of those components in the liver, and particularly by

estrogens. In fact, it was in high temperature females that we found the highest amounts

of PMAs. Being females, estrogens are naturally present in higher amounts than in males,

especially when we think in females at optimal breeding temperature (herein, the highest).

At such temperature more metabolic effort is diverted to reproduction, with implications in

the export of liver lipoproteins to the ovary (Arukwe and Goksøyr 2003) and evoking

hepatocellular remodeling of the liver itself (Rocha et al. 2010). Furthermore, it was

demonstrated in mice that female hormones at physiological concentrations were able to

modulate the phagocytic uptake on macrophages (Chao et al. 1996), and more recent

studies in fish showed the ability of Vtg to enhance the phagocytic abilities of free roaming

macrophages through Fcγ receptor interaction (Liu et al. 2011). These two facts, among

other, may also help explaining proliferation and cellular size increase phenomena

Therefore the higher amount of PMAs in females at both temperatures may be related to

intersex differences in the profile of sex steroids and Vtg levels on serum. On the other

hand, males present less PMAs. Once again, this fact also agrees with the idea of a sex-

37

steroidal regulation of PMAs, as the males naturally have residual amounts of circulating

estrogens and conversely greater levels of androgens, particularly 11-ketotestosterone. It

is worth noting that high temperature males had a higher amount of PMAs, closing in with

the low temperature females. In parallel with other mechanisms, it is possible that in

males at the breeding (high) temperature androgens played a role in modulating PMAs.

We can summarize the trends of the absolute liver volumes of PMAs as follows: low

temperature male < high temperature male < low temperature female < high temperature

female.

In relation to the volume-weighted volume, data are displayed in Table 6 and Figure

14. From the statistical analysis, using ANOVA, we found two different groups (Post-Hoc

Newman-Keuls, p<0.05), one for males and one for females (Figure 14). This data may

additionally show that the size of the PMAs is sex related, and that the higher total volume

of PMAs in females is most likely due to an increase in the number of PMAs; and not due

to an increase of the volume of each PMA. This supports the already described influences

of the sexual endogenous status on the variability and amount of PMAs (Jordanova et al.

2008; Thilakaratne et al. 2007). As the greater values were found in females, whatever the

temperature, the fact support the hypothesis that an estrogenic exposure would be able to

induce the proliferation of PMAs, and consequently an increase in their total volume in the

liver. Only experiments can proof or disproof the hypothesis. On the other hand, as males

had consistently smaller PMAs, we wonder whether androgens may induce such condition.

38

Figure 13 Graphic chart of the absolute volume of PMA vs. experimental group. Three different statistical settings were found (a, b and c; p<0.05). On set a we can find females together while in set c we can only find males. In set b we can find a pair, low temperature female and high temperature male. Box plot graphic with minimum, first quartile, median, third quartile and maximum. Open circles represent statistical outliers (herein not excluded from the analysis).

Table 6 Volume-weighted volume of PMAs in fish acclimated to low and high temperatures.

Volume-weighted Volume (mm³)

Low Temperature

Females High Temperature

Females Low Temperature

Males High Temperature

Males

Mean 2.86x10-4 2.76x10-4 8.07x10-5 9.49x10-5

Standard Deviation 2.49x10-4 2.17x10-4 4.48x10-5 4.60x10-5 Variation Coefficient 0.86 0.78 0.56 0.48

39

Figure 14 Graphic chart of volume-weighted volume of PMAs vs. experimental group. Two statistically significant different sets were found. In set a we can find the females, while in set b we can find the males. Box plot graphic with minimum, first quartile, median, third quartile and maximum. Open circles represent statistical outliers (herein not excluded from the analysis).

Considering the baseline data, and with the aim to start exploring the modulation

and toxicant effects of xenoestrogenic and xenoandrogenic over PMAs, we reasoned that

we should start the experiments with males kept at low temperature. This because males:

a) at low temperature likely have the lowest possible sex-related endocrine influences of

PMAs, especially in relation to estrogenic actions; b) presented low interanimal variability;

and c) tented to have the lowest baseline relative and total values of volumes of PMAs (so

it should be easier, in theory, to detect any toxicant-induced increases of the PMAs).

40

Enzymatic histochemistry assay

With a basic analysis on the color saturation using imaging processing software, we

were able to compare the four groups. The PMAs presented different color intensities from

group from group as it can be seen at Table 7 and Figure 15.

Table 7 Color saturation of PMAs histochemically stained for acid phosphatase.

Color Saturation (%)

Animal Low Temp. Male Low Temp. Female High Temp. Male High Temp. Female

1 32 49 65 49

2 30 55 40 47

3 36 57 49 47

4 48 50 53 39

5 48 50 n.a 38

Mean 39 52 52 44

Standard Deviation 9 4 10 5 Variation Coefficient 0.23 0.07 0.19 0.11

In fish kept at lower temperature we found a higher saturation of color in the females

than that in males (t-test, p<0.05). As increased color saturation means more processed

substrate, we can argue that low temperature females displayed more AP activity/content

than low temperature males. This was expected as it could be suspected that an increase

on PMAs size would be followed by an increased activity/content in enzymes involved in

phagocytic processing, like the AP. On the higher temperature we found that males and

females did not differ statistically, despite the females tended to have higher liver contents

of PMAs. This difference may be due to a negative correlation between AP activity and

estrogenic hormones, as reported for instance for mice serum AP (Rahnama et al. 2002).

Also curious is the variability of the data, which is lower in females. This may be just

casual or else it indicates that AP activity/content in the female liver PMAs are under more

strict regulation by endogenous factors. This may make some sense if we think that the

fish females had greater hepatic remodeling events compared to males; despite this latter

phenomenon is better studied only in seasonal fish breeders (Rocha et al. 2010). In a

toxicological context, such issues should be taken into account, namely when using AP

activity changes in the PMAs as biomarkers (Broeg 2003).

41

Figure 15 Liver sections histochemically stained for AP. On a and b we can actually see how the low temperature female (b) has more color saturation than the low temperature male (a). On the other hand, the high temperature male (c) presents more saturation than the same temperature female (d). Arrows point to PMAs.

Exposure to ethynylestradiol

During the assay only one (and exposed) animal died. The mean temperatures were

22.3 ± 0.7ºC for the solvent control and 22.2 ± 0.5ºC for the exposed animals. The data

retrieved at sampling is shown in Tables 8, 9 and 10. There were no statistically significant

differences as to gonad weight, total length or standard length. This follows the results of

the first baseline test with the temperature challenge, where the lengths and gonad weight

were similar between experimental groups for the same sex. However, and despite fish

were being initially assigned randomly either to the solvent or to the EE2 aquarium, after

exposure period, males exposed to EE2 were heavier and longer than the control ones (t-

test, p<0.05). Despite this, both groups did not differ in relation to both liver and testis

related parameters (t-test, p>0.05).

42

The finding that platyfish males were heavier and lengthier after exposure to EE2 is

not clear. Theoretically, it would be possible that the simple random assignment that was

made at the start of the experiment failed to promote two equally distributed groups. This

hypothesis is unlikely, namely because not only a random assignment is a sound method

but also after the distribution we made a careful visual inspection to seek for imbalances.

On the other hand, as it is known that in fish estrogens play a role in aspects related with

weight, despite differently according to the species and conditions, another hypothesis to

explain a weight increase would be that EE2 induced some water or lipid accumulation,

namely in muscle. However, the latter hypothesis would not explain increases in lengths.

A growth promoting effect of EE2 at the concentrations used could explain both an increase

in weight as in length. In fact, there is evidence that this may actually happen in fish, as for

example well shown by the fact that E2 treatment for 3 months stimulated an increase in

length and weight of juvenile male and female perch (Perca flavescens), when compared

to control animals (Goetz et al. 2009); the underlying mechanism seems to be shifts in the

liver metabolism towards the production of lipoproteins and carbohydrate binding proteins.

On the other hand, there is also some literature that states otherwise (Shved et al. 2008),

describing negative effects on male growth after exposures to EE2, even at environmental

relevant concentrations. But neither of these studies was conducted on platyfish or other

tight related fish, and so we may speculate that EE2 may have different effects on growth

on different fish families; and naturally also when fish are exposed to different amounts.

43

Table 8 Body, liver and gonad related gross measurements of control male fish from the ethynylestradiol assay


Standard Total Total Weight (g) Total Weight (g) Half Weight for MO (g) Remaining Weight (g) Total Weight (g)

Solvent Control


Table 9 Body, liver and gonad related gross measurements of exposed male fish from the ethynylestradiol assay


Standard Total Total Weight (g) Total Weight (g) Half Weight for ME (g) Remaining Weight (g) Total Weight (g)

Etinyl-Estradiol 75ng/L


Table 10 HSI and GSI data and statistical test related to fish exposed

Index Index t-test (p value)

HSI GSI HIS GSI HSI GSI

Solvent Control Etinyl-Estradiol 75ng/L

Mean 0.023 0.011 Mean 0.020 0.010 0.076 0.254 Standard Deviation 0.007 0.003 Standard Deviation 0.002 0.002

Variation Coefficient 0.28 0.26 Variation Coefficient 0.12 0.20

44

The data collected from the stereological study can be found in Table 11. Once

again, no statistically significant differences were found (p>0.05). One important aspect is

that the variability of the final data was quite high, in both experimental groups. This is well

in line with the data from the experience studying temperature and sex effects. This effect

may blur the use of this parameter in biomarker approaches, and suggests that for such

studies a small sample should not easily reveal differences, at least subtle ones.

Table 11 Table with values estimated for liver volume, relative PMA volume and absolute PMA volume.

Solvent Control Ethynylestradiol 75ng/L

Liver Volume (mm3)

Mean 7.749 8.892 Standard Deviation 3.636 2.592

Variation Coefficient 0.469 0.292

Relative Volume PMA (VV)

Mean 0.16% 0.23% Standard Deviation 0.16% 0.13%


Absolute Volume PMA (mm3)

Mean 0.012 0.020 Standard Deviation 0.008 0.016


Finally, we measured the PMA volume-weighted volume on the solvent control and

exposed animals, as it was done with the first animals acclimated to either low or high

temperatures. The data can be seen at Table 12. Despite the systematic suggestive trend

towards higher mean values in the exposed animals, no statistically significant differences

existed between groups (p>0.05). The variability was extremely high for the solvent group.

This was primarily caused by some larger PMA, which pulled the mean value up, but they

are not representative of the majority of the PMA. In an attempt to have a better illustrate

the values of the majority, we looked into de median values, which can be found at Table

13. From the related statistical analysis, no significant difference was found between the

two groups (p>0.05). Anyway, the exposed animals appear to reveal larger PMAs than the

control ones. This may indicate that EE2 had some sort of effect, but it had not enough

time or input to be significant. This follows the kinetics for EE2 induced Vtg expression

45

(Schultz et al. 2001). The Vtg expression suffers a lag of about 12h before serum Vtg

levels start to rise, which only achieve their maximum at 7th to 9th day. Maybe, this leaves

less time for the possible EE2 and Vtg roles on PMA activation and proliferation (Liu et al.

2011). Furthermore, molecular and cellular remodeling takes less time and afford than all

tissue remodeling to happen. Taking these subjects onto consideration, we may speculate

that the exposed fish were not given enough time to express a quantitative, and significant

difference, and that new studies with more input and/or time could offer positive results.

Table 12 Data for the volume-weighted volume of PMAs in the fish analyzed from the second assay. Each value of a fish represents the mean obtained for that animal.

Mean Volume (mm³)

Solvent Control Ethynylestradiol 75ng/L

1 5.92x10-5 1.56x10-4

2 1.13x10-4 1.01x10-4

3 1.74x10-4 1.07x10-4

4 5.70x10-4 2.79x10-4

5 6.41x10-5 2.33x10-4

Mean 1.96x10-4 1.75x10-4

Standard Deviation 2.14x10-4 7.83X10-5


Table 13 Data for the collected from volume-weighted volume PMAs in the fish analyzed from the second assay. Each value of a fish represents the median obtained for that animal.

Median Volume (mm³)

Solvent Control Ethynylestradiol75ng/L

1 3.42x10-5 5.67x10-5

2 6.41x10-5 3.87x10-5

3 4.02x10-5 7.71x10-5

4 1.63x10-4 1.23x10-4

5 2.48x10-5 6.94x10-5

Mean 6.54x10-5 7.30x10-5

Standard Deviation 5.67x10-5 3.15x10-5


46

Enzymatic histochemistry assay

Like in the first assay, basic color saturation was measured on the cryosections. The

data can be seen at Table 14.

Table 14 Color saturation of AP staining in PM, from fish that experienced the second assay.

Color Intensity (%)

Animal Solvent Control Ethynylestradiol 75ng/L

1 41 32

2 37 25

3 41 21

4 42 35

5 68 n.a

Mean 40 28

Standard Deviation 2 6


We found bigger color saturation on solvent control animals than those in exposed

animals (p>0.05, Figure 16). This means that exposed animals to EE2 had less AP

activity. This data are in line both with the first trial and also with the results documented

by Rahnama (2002), in a study where estrogenic compounds have an inhibitory effect on

AP activity.

Figure 16 Liver sections stained with AP specific enzymatic staining. On a we can see a liver section of a solvent control animal. In b we can see a liver section of an exposed animal. Note the more vivid staining at PMA in a (arrow) when compared with that in b.

47

Immunhistochemistry assay

The results of Immunohistochemistry can be seen in Figure 22. The method was

tested before and the positive control show us that the antibody worked on the zebrafish

(Figure 17, a). Despite this, both solvent control and exposed animals did not mark with

this staining (Figure 17, b and c). This may be due to two different things: 1) there is no

Vtg expression, or 2) the primary antibody does not work for platyfish liver. Knowing that

exposure to EE2 causes Vtg expression (Schultz et al. 2001), and analyzing H&E slides

(Figure 18) from both fishes, we may conclude it is due to lack of affinity of the primary

antibody used on technique, as there is an increase in the tissue basophilia, which can be

attributed to an increase on rough endoplasmatic reticulum (RER) on exposed animals.

Eosinophilia of cytoplasm

We also looked into the H&E liver slides and evaluated its degree of eosinophilia or

basophilia. The more cytoplasmatic contents the cell has, namely in mitochondria and

smooth endoplasmic reticulum, by one hand, and in RER, by other hand, the greater the

affinity for either eosin or for hematoxylin, respectively. Intermediate tones (from deep pink

to bluish mean) a balance of organelle contents and consequent mixed staining tinges.

RER in particular is expected to be more present on the exposed fishes as the result of

the increased Vtg expression. In fact, this phenomenon was verified. This was supported

by electron microscopy analysis — as made in other parallel work with the same animals

and where an increase on RER is seen (Figueiredo 2011). Light microscopy images also

support this (Figure 18). The exposed fishes have a denser and more strongly tinged (red-

bluish) cytoplasm, thus supporting that EE2 induced an increase of Vtg production.

48

Figure 17 Liver slides stained for vitellogenin, with specific immunohistochemistry. a is a positive control to the method, using zebrafish liver. b is a liver section of a solvent control platyfish. c is a liver section of an exposed platyfish. As seen in a, the method did work, has the hepatocyte cytoplasm is positive for vtg. In the platyfish liver (b and c), the staining was negative apart from some background staining.

Figure 18 Liver section stained with H&E. In a we have a solvent control animal, with no exposure to EE2. On b we have an exposed animal which was exposed to 75ng/L of EE2. Note the difference in the cytoplasm at b, with the cells presenting a more heavily (more purple) stained on the exposed animal.

49

Conclusion

In this study we tested and worked on an unbiased quantitative method to evaluate

volume-related changes in PMAs on a histopathological level, to be used on experimental

assays and ultimately on biomonitoring assays. With the results obtained from the several

tests, regarding conservation and staining, we conclude that best results were achieved

using Bouin’s fixative and PAS staining protocol. Together with the proper stereological

procedures used – by using differential point counting, point-sampled intercepts, and the

Cavalieri estimator, we implemented a theoretically sound, straightforward and relatively

fast method for quantifying changes in PMAs on a histopathological level. We also found

that the platyfish liver PMAs were modulated by the fish sex indicating to some extent a

possible endogenous hormonal control. Whatever the tested temperatures, females had

consistently higher amounts of PMAs, which were also bigger than those of males. Finally,

from the sub-chronic exposure of males we found that PMAs were unresponsive to EE2,

at least at the dosage and time of exposure tested. This aspect despite not refuting the

possibility of a sex-steroid modulation of PMAs, at least is a warning for the use of PMAs

as an additional biomarker of xenoestrogenic (and eventually other toxicants) pollution in

subacute situations. Also, from the exposure assay, we found that the activity of AP in the

PMAs showed signs of a reduction when animals were exposed to EE2. In terms of our

operational objectives, we: 1) established and implemented an unbiased stereological

method to evaluate histopathological changes in the amount of fish liver PMAs for use

both in experimental assays and in biomonitoring; 2) discovered the fish sex, but not the

temperature (breeding optimal versus lower non-optimal), had a consistent effect on the

baseline liver content of PMAs; and 3) could not prove with a first experimental approach

that the amount/activity of fish liver PMAs in directly influenced by subacute exposure to

EE2. Also, the study warned that using the amount of PMAs without actually knowing and

controlling at least the effect of the sex will likely biases related biomarker approaches.

The data calls for further experimental studies, namely playing the variables in question,

for example conducting chronic exposures at environmentally relevant concentrations.

50

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