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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, Brazil
b
Post-Graduation Program on Science Materials – PGCIMAT, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, CEP 91501-970 Porto Alegre, RS, Brazil
c
Oceanographic Institute, University of São Paulo, Praça do Oceanográfico, 191 Butantã, CEP 05508-120 São Paulo, SP, Brazil
article info
Article history:
Received 14 April 2013
Received in revised form 20 May 2013
Accepted 25 May 2013
Available online 17 August 2013
Keywords:
Micro-PIXE
PIXE
Injuries in fish liver
abstract
Fish liver is the primary organ related to the biotransformation of organic contaminants and metals. This
organ is very sensitive to organic and inorganic contaminants and can accumulate them in higher
amounts 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 of
the tissue and usually accompanied by pigmented cells. The aim of this study is to apply micro-PIXE in
combination with conventional PIXE as a qualitative and quantitative analysis of elements to characterize
histopathologies 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 of
these elements, suggesting the presence of melanomacrophage centers which are too small to be
detected by histopathological conventional methods. Broad PIXE results showed that the concentration
of 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 not
to melanomacrophage centers.
Ó2013 Elsevier B.V. All rights reserved.
1. Introduction
Coastal environments have been transformed by human activ-
ity, especially with increasing addition of substances in marine
and estuarine environments characterizing contamination [1].
Fish can be used as monitors of environmental quality, both, in
terms of the biodiversity of species in the community [2], as well
as 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 important
tool 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, since
certain injuries may be derived from different sources. Therefore,
the diagnostics is not reliable and useful for resolving questions
about the causative agents of injuries [1].
The liver is considered the first organ to identify histological
biomarkers due to its central role in many metabolic functions like
protein 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 of
magnitude 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 chronic
inflammatory injuries and cell degeneration. The MMCs are clus-
ters of monocytes containing melanosomes, lysosomes, and an
accumulation of ceroid and lipofuscin. Several studies have
demonstrated positive correlations between liver injuries and
bottom-living fishes exposed to contaminated areas [5].
As histological studies are time-consuming and present low
specificity, the present study was focused on using a new tool to
evaluate modifications on micro-structures of fish liver with mi-
cro-PIXE along with broad PIXE as techniques to determine the
presence 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 Ó2013 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, Federal
University of Rio Grande do Sul, Av. Bento Gonçalves 9500, P.O. Box 15051, CEP
91501-970 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
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B
journal homepage: www.elsevier.com/locate/nimb
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]. This
enables a deep analysis on which elements are present on healthy
tissues, on MMCs and on other organ alterations.
2. Materials and methods
Specimens used in this study were caught in the Santos-São
Vicente estuarine system, located in a tropical area of Southeastern
Fig. 1. Photomicrography of fish liver. Panels A–D represent liver tissues in the
following conditions: (A) healthy liver with its morphology preserved and without
apparent injuries or MMCs; (B) liver with injuries with MMCs (red arrows), showing
disorganization and small focus steatosis (green arrows); (C) liver with injuries and
no apparent MMCs, showing small focus of necrosis (blue arrows) and steatosis
(green arrows); (D) liver with injuries but without apparent MMCs, showing focus
of steatosis (green arrows). (For interpretation of the references to colour in this
figure 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. The
scanned area is 1500 1500
l
m
2
. The panels show liver tissues in the following
condition: (A) healthy liver; (B) liver with injuries and MMCs; (C) liver with injuries
but no apparent MMCs as revealed by the histopathological study. The MMCs both
in 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 by
several 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 to
their liver condition classified as follows: healthy liver tissue; in-
jured liver tissue with MMCs; and injured liver tissue without
MMCs. From each case, three fish specimens were chosen and, of
those, 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 order
to obtain elemental maps. On the other hand, PIXE measurements
of all eighteen samples were performed for the quantitative
analysis.
2.1. Sample preparation
For the histopathological characterization, well known proto-
cols were used. The livers were extracted right after the fish’s death
and were fixed on a 10% buffered formalin solution. Then, the livers
were dehydrated on crescent alcoholic solutions from 70% to 100%
for 60 min. Subsequently, they were submitted to diafanization in
two xylene baths of 60 min and then embedded in paraffin. After
cooling down, the blocks were placed in a microtome and 4–
5
l
m thick liver slices were produced. Finally, they were mounted
on glass slides and dyed with hematoxylin/eosin for light micros-
copy analysis.
For the analysis of fish liver by micro-PIXE technique, some
modifications in the standard protocol had to be made. First of
all, 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 such
problems. In this case, a 2
l
m thick Mylar
Ò
substrate was used
for fixing the fish tissue.
Another problem on the traditional histopathological analysis is
the use of dyes that may contain metals in their composition which
could show up in the X-ray spectra. Because of that, the samples
for PIXE and micro-PIXE analyses were not dyed. However, tissue
modifications without dye are very difficult to be observed under
an optical microscope. To solve this problem, a two-step process
was devised. Initially, a tissue slice was cut from the paraffin block,
fixed in a glass slide, and finally dyed. The stained tissue injuries
were located under an optical microscope and mapped in the glass
slide. 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 fixed
on Mylar
Ò
substrates.
2.2. Micro-PIXE measurements
Micro-PIXE measurements were performed using the Oxford
Microbeams
Ò
system operating in triplet mode. The proton beam
current varied between 20 and 150 pA while the beam energy
was kept fixed at 3 MeV. The spot size was about 2.5 2.5
l
m
2
.
Measurements were performed with different scan sizes, from
75 75
l
m
2
to 1000 1000
l
m
2
. X-rays induced in the samples
by the proton beam were detected with a Si(Li) detector placed a
135°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 is
300 300
l
m
2
. 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 were
obtained for several tissues.
2.3. PIXE measurements
For quantitative analysis, PIXE measurements were performed
with 2.0 MeV proton beam. Typical beam currents were about
1 nA. The beam spot size of 1 1cm
2
was large enough to cover
the entire sample. X-rays were detected by a Si(Li) detector with
an energy resolution of 155 eV at 5.9 keV. The detector was placed
at 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 analyses
were performed as well using ANOVA One Way and Tukey’s post
hoc tests in order to check differences among the groups.
3. Results and discussion
Fig. 1 shows the histopathological analysis of fish liver showing
a health liver, an injured liver without melanomacrophage centers
(MMCs) and an injured liver with MMCs. A 1500 1500
l
m
2
scan
of the samples showed that there are no irregularities on the iron
concentration of healthy tissue samples (Fig. 2). On the other hand,
liver samples that presented melanomacrophage centers on the
histopathological study showed iron spots on the micro-PIXE anal-
ysis. Moreover, samples with injuries but without apparent mela-
nomacrophage centers on the histopathological study presented
smaller 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
l
m
2
) of a mela-
nomacrophage center. The elemental maps reveal that clorine is
homogeneously distributed over the tissue, while elements such
as phosphorus and sulfur are a little more concentrated in the
MMC. Iron occurred in the MMC with higher concentrations in
comparison to other metals like copper, zinc and nickel, suggesting
that 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, zinc
and nickel, a much longer measurement would have to be made to
achieve enough statistics to produce better elemental. However,
our results indicate clearly that these elements correlate with the
MMC. Although some metals like iron, copper, zinc and cobalt
are considered essential to biological processes, they can be toxic
when present in higher concentrations [9].
Concerning the samples containing smaller MMCs, scans of
150 150
l
m
2
were performed in order to obtain more detailed
information of these structures. According to the results shown
in Fig. 4, the concentration of iron is much higher in the MMCs.
Moreover, a faint but still visible correlation between the MMCs
and copper, zinc and to a lesser extent nickel was observed.
Results of the broad PIXE analyses can be seen in Fig. 5. As can
be seen, some elements like Ti and Fe have higher concentrations
in the injured tissue with bigger MMCs. This suggests that these
elements 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 is
150 150
l
m
2
. 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 elements
are related to the injury rather than with the MMCs. However, it
must be stressed that from the statistical point of view, no signif-
icant differences were observed for most of the elements among
the groups. Further studies with larger number of samples are
needed in order to have a better picture relating the elements with
the injuries studied in this work.
4. Conclusions
In this work we performed the elemental characterization of
fish liver tissues using PIXE and micro-PIXE techniques. The results
show that melanomacrophage centers (MMCs) are rich in Fe, while
other metals like Ni, Cu and Zn appear in smaller concentrations.
Elements like Ti and Fe appear to correlate with the presence of
MMCs, while Mn and Ni could be related to the injuries them-
selves. However, no significant differences were observed for most
elements from injured tissues with MMCs visible or not under his-
tological analyses. Further studies could shed some light in this
issue.
In spite of being a sensitive tool to diagnose toxic effects that af-
fect animal tissues, the routine histological techniques are limited
and cannot provide further details of lesions generated in tissues as
a biological response to injury and stress. However, when associ-
ated to other methods of analysis such as PIXE and micro-PIXE, a
comprehensive understanding of lesions in organic tissues can be
achieved.
Acknowledgements
The authors acknowledge the financial support from CAPES
(Nanobiotec Brasil Program) and CNPq. Moreover, we are indebted
to 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 bars
stand for injured tissues with MMCs visible and not visible by routine histopathological analyses, respectively.
E.M. Stori et al. / Nuclear Instruments and Methods in Physics Research B 318 (2014) 83–87 87
