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Biotechnic & Histochemistry
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A histopathological and stereological study of liver
damage in female rats caused by mercury vapor
A Yahyazedeh, BZ Altunkaynak, N Akgül & HM Akgül
To cite this article:
A Yahyazedeh, BZ Altunkaynak, N Akgül & HM Akgül (2017) A
histopathological and stereological study of liver damage in female rats caused by mercury vapor,
Biotechnic & Histochemistry, 92:5, 338-346, DOI: 10.1080/10520295.2017.1312527
To link to this article: https://doi.org/10.1080/10520295.2017.1312527
Published online: 09 Jun 2017.
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A histopathological and stereological study of liver
damage in female rats caused by mercury vapor
A Yahyazedeh
1, BZ Altunkaynak
1, N Akgül
2, HM Akgül
31
Department of Histology and Embryology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, and, Departments of 2
Restorative Dentistry, and3Oral Diagnosis and Radiology, Faculty of Dentistry, Atatürk University, Erzurum, Turkey Accepted March 25, 2017
Abstract
We examined the possible effects of elemental mercury vapor on the liver of the female rats. We
divided the animals into an untreated control group and an experimental group that was
exposed to mercury vapor for 45 days. Liver samples were obtained for histological and
stereo-logical analysis. The total liver, parenchyma and sinusoid volumes were increased signi
ficantly
in the mercury vapor treated group compared to controls. Also, the mean density, total number
and mean nuclear diameter of hepatocytes, except for binucleated hepatocytes, was decreased in
the experimental group compared to controls. Light and electron microscopy revealed alterations
of liver structure of the experimental animals compared to controls.
Key words: electron microscopy, histology, liver, mercury vapor, rat, stereology, toxicity
Mercury (Hg) is an environmental pollutant that
is toxic to humans (Selin
2009
). Mercury vapor is
the inhaled form of elemental Hg (Björkman
et al.
2007
, WHO
2003
). The vapor can be formed
from dental amalgam, liquid metallic Hg, Hg
compounds, and mines. Hg is lipophilic and is
absorbed easily by the oral mucosa, skin and
lungs. After entering the blood, mercury vapor
is oxidized to toxic divalent inorganic Hg
(Clarkson
1997
), which accumulates in various
tissues (Björkman et al.
2007
, Iranmanesh et al.
2013
, Yasutake et al.
2010
). Divalent inorganic Hg
stimulates production of reactive oxygen species
(ROS) and ROS reduce the level of antioxidant
enzymes (Goering et al.
2002
, Zhao et al.
2009
).
Also, after absorption by the body, mercury
vapor causes tissue damage by increasing
intra-cellular calcium storage and in
flux of calcium
from the extracellular
fluid. The deleterious
effects of ROS on mitochondrial membrane lipids
and proteins initiate the apoptotic cascade (Sunja
et al.
2010
).
Mercury vapor can cause neurological and
behavioral disorders with symptoms including
tremors, memory loss, neuromuscular effects,
and cognitive and motor dysfunction (WHO
1991
). It has been reported that Hg vapor
released from amalgam for
filling teeth
predis-poses people to Alzheimer’s disease (Pendergrass
et al.
1997
). Mercury exposure exerts adverse
effects on genes (Liu et al.
2003
), kidney
(Tanaka et al.
1998
, Akgül et al.
2016
),
cardiovas-cular system (Fernandes Azevedo et al.
2012
),
beta cells of the pancreas (Chen et al.
2010
),
female
reproductive
system
(Apostoli
and
Catalani
2011
, Altunkaynak et al.
2016
) male
reproductive systems (Boujbiha et al.
2009
,
Altunkaynak et al.
2015
) and neurons (Tang and
Li
2006
). Accumulation of Hg decreases
hepato-cyte viability in the liver (Ashour et al.
1993
, Cao
et al.
2012
, Endo et al.
2013
, Karapehlivan et al.
2014
). To clarify the toxicity of mercury vapor
further, we investigated its effects on hepatic
architecture using quantitative methods.
Correspondence: B. Zuhal Altunkaynak, Faculty of Medicine, Department of Histology and Embryology, Ondokuz Mayıs University, Samsun, Turkey. E-mail:berrinzuhal@gmail.com
Color versions of one or more of thefigures in the article can be found online atwww.tandfonline.com/ibih
© 2017 The Biological Stain Commission
Material and methods
Experimental animals and procedure
Our investigation was conducted with the approval of
the
Experimental
Research
and
Application
Committee of the Ataturk University, Erzurum,
Turkey. We used 10
−12-week-old 200 g female
Wistar albino rats. Rats were obtained from the
Experimental Animal Research and Application
Center of Atatürk University. The animals were kept
at 22 ± 2° C with optimal humidity and a 12 h light:12
h dark cycle. The rats had access to food and water ad
libitum. We divided the animals into control and
experimental groups, each with six rats. The
experi-mental group was exposed to 1 mg/m
3/day mercury
vapor by inhalation for 45 days within a special
cham-ber as described in our earlier report (Altunkaynak
et al.
2015
). The untreated controls were housed for 45
days in an identical chamber without exposure to Hg
vapor. After exposure to Hg, all rats were
anesthe-tized by inhalation of 2
−3% sevoflurane (Sevorane®
Liquid 250 ml; Abbott, Istanbul, Turkey) in 100%
oxy-gen. After sacri
fice and perfusion with 4%
parafor-maldehyde, the liver was removed from each rat.
Histology
After removal, the livers were cut into small pieces
by systematic random sampling (Altunkaynak and
Ozbek
2009
) and
fixed in 10% formaldehyde. Also,
some pieces were placed in glutaraldehyde for
elec-tron microscopy. For light microscopy, liver samples
were dehydrated through an ascending series of
alcohols, cleared three times in xylene, then
embedded in fresh paraf
fin. Light microscopy
sec-tions were cut at 5
μm using a rotary microtome
(Leica RM 2225; Leica Instruments, Nussloch,
Germany). The sections were placed on glass slides
and stained with hematoxylin and eosin for light
microscopy (Kiernan
2008
, Guven et al.
2013
). These
slides also were used for stereological analyses.
Stereology
Volumes of total liver, sinusoids and parenchyma
We used the Cavalieri method (Dursun et al.
2010
,
Altunkaynak et al.
2012a
) to estimate liver volume
(Altunkaynak et al.
2012a
, Arslan et al.
2016
). A pilot
study was performed to determine the suitable point
density for the point counting grid. Selecting the
appropriate grid size provides an acceptable coef
fi-cient of error (CE) for the area of interest in each
section (Bulut et al.
2014
, Khoshvakhti et al.
2015
).
The CE and coef
ficient of variation (CV) were
estimated to determine whether the point density of
the point counting grid and sectioning intervals were
appropriate (Göçer et al.
2016
). According to
pre-vious studies, CE values <0.05 are sufficient to ensure
reliability of the stereological studies. CE and CV
values were estimated using the formulas below.
The volumetric values [V(total)] of interest were
esti-mated using the formula (Uyanık et al.
2009
):
V total
ð
Þ ¼ t a p
P
where t is the thickness of the section, a(p) is the
interval point area and
ΣP is the total number of
points that touch appropriate areas in the sections.
Mean numerical density, and number of
hepatocytes and binucleated hepatocytes
We used the physical disector method (Altunkaynak
et al.
2011a
) to obtain unbiased numerical data.
Selection of the physical dissector pairs was
per-formed as described by Sterio (
1984
). The
first section
selected (reference section) and its adjacent section
(look up section) are de
fined as a dissector pair.
These sections were separated by 20
μm, which was
the thickness of four sections as a rule of the physical
disector. According to this rule, the distance between
the section pairs must be approximately 30–40% of
the average projected height of the interested object
to be estimated. In this way, approximately 15
–20
section pairs were obtained for evaluation.
For selection, the
first section was selected
ran-domly from the
first four sections, e.g., we selected
the third section. We then systematically continued
sampling by selecting each consecutive fourth section,
i.e., the
first pair = third section and seventh (3 + 4)
sections, the second pair = 11th and 15th (11 + 4)
sections etc. Digital photographs were taken at 4, 10
and 40 × magni
fications for each section. An unbiased
20 × 20 cm
2counting frame (Altunkaynak et al.
2011b
)
was superimposed on the sampled regions of the
digital photographs of the sections for calculation of
the number of hepatocytes. According to the rule of
physical disector, hepatocytes that were observed in
the reference section, but not in the look-up section,
were countable. Initially, the numerical frequency of
both hepatocytes and binucleated hepatocytes were
computed; later the numerical density of only
binu-cleated hepatocytes was counted in similar areas of
the section pairs (Fig. 1c, d).
The mean numerical density of hepatocytes/cm
3was calculated as:
Nv
¼
P
Q
P
Vdisector
where
P
Q
is the total number of hepatocytes and
P
V disector is the total volume of disector frames.
The total number of hepatocytes then was estimated
by multiplying NV in total volume of liver (Vref) as:
Total number of hepatocytes = Nv
∙Vref
We estimated the mean numerical density of
cleated hepatocytes and total number of
binu-cleated hepatocytes using the same approach.
Finally, we estimated the mean diameter of
hepa-tocytes using the equation:
Diameter
¼
P
Q
P
Q
xt
where
∑Q is the total number of nuclei observed in
the reference section,
P
Q
is total number of nuclei
observed in the reference section, but not in the
look-up section, and t is the mean thickness of sections. CE
and CV indicate reliability of the counted cells and
biological variability, respectively (Altunkaynak
et al.
2012b
).
Electron microscopy
Blocks of livers were
fixed in 3% buffered
glutaralde-hyde for 1 h, post
fixed in 1% osmium tetroxide for 90
min, then passed through acetone and propylene
oxide. Samples were embedded in Araldite CY 212
(Agar Chemicals, Tekser Ltd. Sti,
İstanbul, Turkey)
(Harorli et al.
2009
). Semithin sections were cut at 1
µm by an ultramicrotome (LKB NOVA, Bromma,
Sweden) and stained with toluidine blue. These
sections were used to examine the general
organiza-tion of the liver at the light microscopic level. Thin
sections were cut from the same tissue blocks and
stained with uranyl acetate and lead citrate, and used
for ultrastructural examination (Ayranci et al.
2013
,
Çolakoglu et al.
2014
). Sections were viewed using a
Jeol 100 SX electron microscope (Jeol, Tokyo, Japan).
Statistical analysis
We analyzed our data using Mann Whitney-U
statis-tical test (SPSS software, version15.0; IBM Corp,
İstanbul, Turkey). Data are expressed as medians ±
SEM. Values for p
≤ 0.05 were considered significant.
Results
Stereology
The stereology results are summarized in
Table 1
.
The total volume of the liver was increased signi
fi-cantly in the Hg treated group compared to controls.
The volume of the parenchyma was decreased
sig-ni
ficantly, but the volume of the sinusoids was
increased in the Hg treated group compared to
con-trols. In the experimental group, the mean numerical
density and total number of hepatocytes were
reduced signi
ficantly, but the mean numerical
den-sity and total number of binucleated hepatocytes
were increased signi
ficantly in the Hg treated
group compared to controls. The nuclear diameter
Table 1. Morphometric evaluations of control and experimental groups with CE values
Estimation Control CE Hg CE p LV via CM (cm3) 9.98 ± 1.21 0.002 11.095 ± 3.11 0.002 < 0.01 Volume of parenchyma (cm3) 6.70 ± 1.34 0.012 5.840 ± 1.17 0.023 < 0.05 Volume of 3.73 ± 0.87 0.03 5.48 ± 1.7 0.029 < 0.01 sinusoids (cm3) ND of hepatocytes 211.65 × 103± 1428 0.01 169.86 × 103± 1297 0.017 < 0.01 (cell/cm3) MND of 29.93 × 103± 832 0.04 38.79 × 103± 644 0.038 < 0.05 binucleated hepatocytes (cell/cm3) TN of 2.11 × 106± 105613 0.01 1.88 × 106± 94229 0.01 < 0.05 hepatocytes TN of binucleated 298.73 × 103± 14936 0.02 430.43 × 103± 23180 0.032 < 0.01 hepatocytes NH of hepatocytes (μm) 7.90 ± 0.81 0.04 5.16 ± 0.44 0.043 < 0.01
Means ± SEM. LV, liver volume; CM, Cavalieri method; MND, mean numerical density; TN, total number; NH, nuclear diameter; CE, coefficient of error.
of the hepatocytes was decreased significantly in the
group exposed to Hg compared to controls.
Light microscopy
We observed enlarged blood vessels and dilated
sinusoids with increased perivascular connective
tissue in the Hg treated group (
Fig. 2
). We also
found extensive degeneration of hepatocytes in
sec-tions from the experimental group (
Fig. 3
). The
degenerated hepatocyte cytoplasm was stained
darkly and no nucleolus could be found (
Fig. 3
).
The livers of the control group appeared normal
(
Figs. 1a
,
2a
).
Electron microscopy
Electron microscopy of the experimental group
showed mildly to moderately pyknotic nuclei.
Irregular cellular borders, edematous and expanded
spaces of Disse, enlarged bile canaliculi and
sepa-rated adhesion complexes indicated degeneration in
the experimental group. We also observed dilated
rough endoplasmic reticulum and sinusoids,
degen-erated mitochondria and accumulation of electron
dense material. Vacuoles also were evident in some
hepatocytes of the experimental group compared to
normal appearing controls (
Figs. 4
,
5
).
Discussion
The liver degrades or eliminates toxic materials
including Hg, which is a common pollutant in the
environment. Liver tissue is susceptible to injury
from environmental and industrial mercury (Lee
et al.
2014
, Garcia-Niñoand Pedraza-Chaverrí
2014
). Exposure to Hg increases products of lipid
oxidation (Huang et al.
1996
), impairs the function
of mitochondria (Belyaeva et al.
2011
), reduces the
activity of metabolic enzymes, decreases synthesis
of hepatic coagulation factors (Chang et al.
1973
),
interferes with antioxidant enzymes (Pal and Ghosh
2012
) and causes vascular injury (Kanluen and
Gottlieb
1991
). Hg exposure increases oxidative
Fig. 1. a, b) Point counting grid for the Cavalieri principle. c, d) Consecutive pair sections for applying the physical dissector. Images in c) and d) are reference and look-up sections, respectively. *, countable profiles of hepatocytes as disector particle; X, uncountable profiles according to the rule of unbiased counting frame.
Fig. 2. Sections of liver from control (a) and experimental (b, d) groups.−, increased perivascular connective tissue; arrow, dilated sinusoid; +, enlarged blood vessel.
Fig. 3. Semithin sections from liver of control (a, b) and experimental (c−f) groups. −, abnormal intracellular accumula-tions; +, enlarged blood vessel; arrow, degenerated hepatocytes.
stress (Gutierrez et al.
2006
), which may contribute
to its histopathology.
We used stereological methods to estimate the
number of hepatocytes and to measure the volumes
of the parenchyma and sinusoids (Altunkaynak and
Altunkaynak
2007
). Our stereological and
histo-pathological
findings were consistent with each
other. We found that the total volume of the liver
in the Hg treated group was increased signi
ficantly
compared to controls. The increase may be due to
enlarged blood vessels, increased perivascular
con-nective tissue and increased sinusoid volume.
Moderate hyperaemia in the sections of the
experi-mental group re
flected alteration of sinusoid
volume. Exposure to Hg vapor also caused dilation
of the blood vessels. On the other hand, the mean
numerical density and total number of hepatocytes
were reduced in the group exposed to Hg vapor.
The reduced number of hepatocytes was caused by
degeneration and death of these cells. The mean
numerical density and total number of binucleated
hepatocytes were increased signi
ficantly in the Hg
treated group compared to untreated controls. We
concluded that Hg vapor inhalation caused the
pro-liferation of binucleated hepatocytes. Nuclear
height
of
the
hepatocytes
was
signi
ficantly
decreased in the Hg exposed group and the nuclear
diameter
of
hepatocytes
was
signi
ficantly
decreased. We believe that the decrease might be
due to alteration of the genetic material including
pyknotic or heterochromatic changes
(Cebulska-Wasilewska et al.
2005
, Chen et al.
2005
). Further
investigation is required to clarify the impact of Hg
vapor on human health.
Fig. 4. Electron micrographs of liver sections. a−d) Control group. c) Canaliculi; white arrow, smooth endoplasmic reticulum; asterisk, nucleus; black arrow, rough endoplasmic reticulum; double black arrowheads, border between hepatocytes; single black arrowhead, lysosome; single white arrowhead, mitochondria; x, hepatocyte microvillus; plus, endothelial cell; minus, Disse space; arrow with double heads, sinusoid; black circle, erythrocyte. e, f) Hg vapor treated group. Plus with circle, pyknotic nucleus; ce, irregular borders and enlarged bile canaliculi; double black arrowheads, dilated rough endoplasmic reticulum; white triangle, degeneration of mitochondria; white circle, cytoplasmic edema; hexagon, swollen nucleus; circle with black arrowhead, separated adhesion complex. Scale bars = 0.5 µm.
Declaration of interest: The authors report no
con-flicts of interest. The authors alone are responsible
for the content and writing of this paper.
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