Universidade de São Paulo

2014

Elemental characterization of injuries in fish

liver Nuclear Instruments and Methods in Physics Research B v. 318, p. 83-87, 2014http://www.producao.usp.br/handle/BDPI/46441

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Elemental characterization of injuries in fish liver

E.M. Stori a,b,⇑, M.L.C.F. Rocha c, J.F. Dias c, C.E.I. dos Santos a, C.T. de Souza a,b, L Amaral a,b, J.F. Dias a,b

a Ion Implantation Laboratory, Physics Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, P.O. Box 15051, CEP 91501-970 Porto Alegre, RS, Brazilb Post-Graduation Program on Science Materials – PGCIMAT, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, CEP 91501-970 Porto Alegre, RS, Brazilc Oceanographic Institute, University of São Paulo, Praça do Oceanográfico, 191 Butantã, CEP 05508-120 São Paulo, SP, Brazil

a r t i c l e i n f o

Article history:Received 14 April 2013Received in revised form 20 May 2013Accepted 25 May 2013Available online 17 August 2013

Keywords:Micro-PIXEPIXEInjuries in fish liver

a b s t r a c t

Fish liver is the primary organ related to the biotransformation of organic contaminants and metals. Thisorgan is very sensitive to organic and inorganic contaminants and can accumulate them in higheramounts relative to the environment itself and to other organs. One of the most common injuries is a his-topathology called melanomacrophage centers, characterized as modifications of the cellular structure ofthe tissue and usually accompanied by pigmented cells. The aim of this study is to apply micro-PIXE incombination with conventional PIXE as a qualitative and quantitative analysis of elements to characterizehistopathologies in the liver of fishes. Micro-PIXE results show that there is a higher concentration of Fe,P, K, Ti, Cr, Ni, Cu and Zn in melanomacrophage centers. On healthy tissue, the distribution of these ele-ments is homogeneous. In cases where the histopathological study showed injuries without melanomac-rophage centers, the micro-PIXE analysis showed much smaller clusters with higher concentrations ofthese elements, suggesting the presence of melanomacrophage centers which are too small to bedetected by histopathological conventional methods. Broad PIXE results showed that the concentrationof Si, Cl, K, Ti, Fe and Cu are directly related to the presence of melanomacrophage centers. Moreover,it could be observed that the concentration of Cr, Mn and Ni is directly related to the injuries but notto melanomacrophage centers.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Coastal environments have been transformed by human activ-ity, especially with increasing addition of substances in marineand estuarine environments characterizing contamination [1].Fish can be used as monitors of environmental quality, both, interms of the biodiversity of species in the community [2], as wellas the healthiness of individuals [3]. Fish biomarkers are biologi-cal responses measured on organization levels below individual,namely cells, body fluids, tissues or organs. In this way, they pro-vide an indirect measure of exposure and/or effects of contami-nants [4].

Histopathological biomarkers can detect modifications or inju-ries in several tissues and organs and constitute a very importanttool because they present a relatively fast response to the sub-lethal stressors. These biomarkers are highly sensitive and ecolog-ically relevant, but have low specificities to contaminants, sincecertain injuries may be derived from different sources. Therefore,

the diagnostics is not reliable and useful for resolving questionsabout the causative agents of injuries [1].

The liver is considered the first organ to identify histologicalbiomarkers due to its central role in many metabolic functions likeprotein synthesis, gall secretion, metabolites accumulation, intox-ification and detoxification. These functions make the liver bioac-cumulate higher levels of toxic substances up to several orders ofmagnitude higher than the environment itself or even other organs[1]. One of the tissue’s most characteristic alterations are the mel-anomacrophage centers (MMCs), commonly associate with chronicinflammatory injuries and cell degeneration. The MMCs are clus-ters of monocytes containing melanosomes, lysosomes, and anaccumulation of ceroid and lipofuscin. Several studies havedemonstrated positive correlations between liver injuries andbottom-living fishes exposed to contaminated areas [5].

As histological studies are time-consuming and present lowspecificity, the present study was focused on using a new tool toevaluate modifications on micro-structures of fish liver with mi-cro-PIXE along with broad PIXE as techniques to determine thepresence of heavy metals on MMCs and their respective distribu-tion on the liver tissue [6].

The micro-PIXE technique, among other ion beam analysis tech-niques, has some potentialities for environmental studies. Particu-larly in this case, besides the non-destructive character of the

0168-583X/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.nimb.2013.05.109

⇑ Corresponding author at: Ion Implantation Laboratory, Physics Institute, FederalUniversity of Rio Grande do Sul, Av. Bento Gonçalves 9500, P.O. Box 15051, CEP91501-970 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 7248; fax: +55 51 3308 7286.

E-mail address: elistori@gmail.com (E.M. Stori).

Nuclear Instruments and Methods in Physics Research B 318 (2014) 83–87

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analysis, it can indicate in which way some elements are distrib-uted along the modifications. Moreover, the microscopic character-istic of the microprobe together with the capability of scanning

certain areas of the sample open the possibility of correlating dif-ferent elements by producing maps of each one of them [7]. Thisenables a deep analysis on which elements are present on healthytissues, on MMCs and on other organ alterations.

2. Materials and methods

Specimens used in this study were caught in the Santos-SãoVicente estuarine system, located in a tropical area of Southeastern

Fig. 1. Photomicrography of fish liver. Panels A–D represent liver tissues in thefollowing conditions: (A) healthy liver with its morphology preserved and withoutapparent injuries or MMCs; (B) liver with injuries with MMCs (red arrows), showingdisorganization and small focus steatosis (green arrows); (C) liver with injuries andno apparent MMCs, showing small focus of necrosis (blue arrows) and steatosis(green arrows); (D) liver with injuries but without apparent MMCs, showing focusof steatosis (green arrows). (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

Fig. 2. Elemental maps showing the distribution of Fe in the liver tissues. Thescanned area is 1500 � 1500 lm2. The panels show liver tissues in the followingcondition: (A) healthy liver; (B) liver with injuries and MMCs; (C) liver with injuriesbut no apparent MMCs as revealed by the histopathological study. The MMCs bothin panels B and C are indicated with white arrows.

84 E.M. Stori et al. / Nuclear Instruments and Methods in Physics Research B 318 (2014) 83–87

Brazilian coast. This area presents a history of contamination byseveral compounds due to the intense industrial and harbor activ-ities and the disposal of sewage in the estuarine system [8].

For the present study, three cases were selected according totheir liver condition classified as follows: healthy liver tissue; in-jured liver tissue with MMCs; and injured liver tissue withoutMMCs. From each case, three fish specimens were chosen and, ofthose, two liver tissue samples were extracted for analysis, result-ing in 18 samples (of those, 9 liver replicates). In general, micro-PIXE measurements were carried out for a few samples in orderto obtain elemental maps. On the other hand, PIXE measurementsof all eighteen samples were performed for the quantitativeanalysis.

2.1. Sample preparation

For the histopathological characterization, well known proto-cols were used. The livers were extracted right after the fish’s deathand were fixed on a 10% buffered formalin solution. Then, the liverswere dehydrated on crescent alcoholic solutions from 70% to 100%for 60 min. Subsequently, they were submitted to diafanization intwo xylene baths of 60 min and then embedded in paraffin. Aftercooling down, the blocks were placed in a microtome and 4–5 lm thick liver slices were produced. Finally, they were mountedon glass slides and dyed with hematoxylin/eosin for light micros-copy analysis.

For the analysis of fish liver by micro-PIXE technique, somemodifications in the standard protocol had to be made. First ofall, glass slides are undesirable substrates for PIXE analysis because

of the presence of silica and other elements that interfere on the X-ray energy spectra, causing a misinterpretation of the data. There-fore, it was used a polymer as a substrate in order to avoid suchproblems. In this case, a 2 lm thick Mylar� substrate was usedfor fixing the fish tissue.

Another problem on the traditional histopathological analysis isthe use of dyes that may contain metals in their composition whichcould show up in the X-ray spectra. Because of that, the samplesfor PIXE and micro-PIXE analyses were not dyed. However, tissuemodifications without dye are very difficult to be observed underan optical microscope. To solve this problem, a two-step processwas devised. Initially, a tissue slice was cut from the paraffin block,fixed in a glass slide, and finally dyed. The stained tissue injurieswere located under an optical microscope and mapped in the glassslide. Secondly, the injured regions were re-drawn over the paraf-fin block and through the use of a microarrayer, cylinders contain-ing only modified tissue were cut and new tissue slices were fixedon Mylar� substrates.

2.2. Micro-PIXE measurements

Micro-PIXE measurements were performed using the OxfordMicrobeams� system operating in triplet mode. The proton beamcurrent varied between 20 and 150 pA while the beam energywas kept fixed at 3 MeV. The spot size was about 2.5 � 2.5 lm2.Measurements were performed with different scan sizes, from75 � 75 lm2 to 1000 � 1000 lm2. X-rays induced in the samplesby the proton beam were detected with a Si(Li) detector placed a135� with respect to the beam direction. The energy resolution of

Fig. 3. Elemental maps of a MMC depicting the following elements: (A) chlorine; (B) phosphorous; (C) sulfur; (D) iron; (E) copper; (F) zinc; and (G) nickel. The scan area is300 � 300 lm2. The scale bar is relative to all maps.

E.M. Stori et al. / Nuclear Instruments and Methods in Physics Research B 318 (2014) 83–87 85

the X-ray detector was 165 eV at 5.9 keV. Elemental maps wereobtained for several tissues.

2.3. PIXE measurements

For quantitative analysis, PIXE measurements were performedwith 2.0 MeV proton beam. Typical beam currents were about1 nA. The beam spot size of 1 � 1 cm2 was large enough to coverthe entire sample. X-rays were detected by a Si(Li) detector withan energy resolution of 155 eV at 5.9 keV. The detector was placedat 135� with respect to the beam direction. The quantitative anal-ysis of the X-ray spectra was carried out with the GUPIXWIN soft-ware in the thin target approximation. Finally, statistical analyseswere performed as well using ANOVA One Way and Tukey’s posthoc tests in order to check differences among the groups.

3. Results and discussion

Fig. 1 shows the histopathological analysis of fish liver showinga health liver, an injured liver without melanomacrophage centers(MMCs) and an injured liver with MMCs. A 1500 � 1500 lm2 scanof the samples showed that there are no irregularities on the ironconcentration of healthy tissue samples (Fig. 2). On the other hand,liver samples that presented melanomacrophage centers on thehistopathological study showed iron spots on the micro-PIXE anal-ysis. Moreover, samples with injuries but without apparent mela-nomacrophage centers on the histopathological study presentedsmaller spots of iron, suggesting that despite they were not detect-

able by the light microscope, the samples presented small melano-macrophage centers.

Fig. 3 depicts a more detailed scan (300 � 300 lm2) of a mela-nomacrophage center. The elemental maps reveal that clorine ishomogeneously distributed over the tissue, while elements suchas phosphorus and sulfur are a little more concentrated in theMMC. Iron occurred in the MMC with higher concentrations incomparison to other metals like copper, zinc and nickel, suggestingthat larger amounts of metals are indeed located in this structure.As iron is an essential erythrocytes component, the higher concen-tration can be expected in such structures. Concerning copper, zincand nickel, a much longer measurement would have to be made toachieve enough statistics to produce better elemental. However,our results indicate clearly that these elements correlate with theMMC. Although some metals like iron, copper, zinc and cobaltare considered essential to biological processes, they can be toxicwhen present in higher concentrations [9].

Concerning the samples containing smaller MMCs, scans of150 � 150 lm2 were performed in order to obtain more detailedinformation of these structures. According to the results shownin Fig. 4, the concentration of iron is much higher in the MMCs.Moreover, a faint but still visible correlation between the MMCsand copper, zinc and to a lesser extent nickel was observed.

Results of the broad PIXE analyses can be seen in Fig. 5. As canbe seen, some elements like Ti and Fe have higher concentrationsin the injured tissue with bigger MMCs. This suggests that theseelements are correlated to the MMCs rather than to the injury it-self. On the other hand, elements such as Cr, Mn and Ni present lar-ger concentrations in the liver tissue with small MMCs (not visible

Fig. 4. Elemental maps of a MMC depicting the following elements: (A) chlorine; (B) phosphorous; (C) sulfur; (D) iron; (E) copper; (F) zinc; and (G) nickel. The scan area is150 � 150 lm2. The scale bar is relative to all maps.

86 E.M. Stori et al. / Nuclear Instruments and Methods in Physics Research B 318 (2014) 83–87

in the histopathological analysis), suggesting that these elementsare related to the injury rather than with the MMCs. However, itmust be stressed that from the statistical point of view, no signif-icant differences were observed for most of the elements amongthe groups. Further studies with larger number of samples areneeded in order to have a better picture relating the elements withthe injuries studied in this work.

4. Conclusions

In this work we performed the elemental characterization offish liver tissues using PIXE and micro-PIXE techniques. The resultsshow that melanomacrophage centers (MMCs) are rich in Fe, whileother metals like Ni, Cu and Zn appear in smaller concentrations.Elements like Ti and Fe appear to correlate with the presence ofMMCs, while Mn and Ni could be related to the injuries them-selves. However, no significant differences were observed for mostelements from injured tissues with MMCs visible or not under his-tological analyses. Further studies could shed some light in thisissue.

In spite of being a sensitive tool to diagnose toxic effects that af-fect animal tissues, the routine histological techniques are limitedand cannot provide further details of lesions generated in tissues asa biological response to injury and stress. However, when associ-ated to other methods of analysis such as PIXE and micro-PIXE, acomprehensive understanding of lesions in organic tissues can beachieved.

Acknowledgements

The authors acknowledge the financial support from CAPES(Nanobiotec Brasil Program) and CNPq. Moreover, we are indebtedto Prof. Edna Kimura and M.Sc. Cesar Fuziwara (Biomedical Sci-ences Institute- USP) and Vet. Helcy Silbiger (Oceanographic Insti-tute-USP).

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Fig. 5. Concentration of the main elements present on the liver samples detected by broad PIXE. Light gray bars stand for the healthy liver, while white and dark gray barsstand for injured tissues with MMCs visible and not visible by routine histopathological analyses, respectively.

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