Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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RENAL HISTOPATHOLOGICAL AND MCP-1 MODULATION IN MALE
RAT (Rattus norvigicaus) FOLLOWING REPEATED TiO2 NPs
INTRATRACHEAL INSTILLATION
Fatimah A. Jasim*, Dhamia K. Suker**
* Biology Instructor, The Gifted Students' School- Basrah, Ministry of Education, Iraq
** Cell, and Biotechnology Research Unit , College of Science ,University of Basrah , Iraq.
Keywords: Renal histopathology, IL-10 alteration, BALF proteins.
Corresponding author E.mail: fjasim119@gmail.com
ABSTRACT
The rapidly expanding field of nanotechnology is becoming a possible source for human
exposure to nanoparticles. Titanium dioxide nanoparticles (TiO2-NPs) is one of an important
nanoparticle which has been widely manufactured in developed processes for several years. The
aim of this study was to investigate the effects of TiO2-NPs on some biochemical parameters and
the renal histopathology alterations in adult Wistar rats. 63 male rats were used and treated with
different doses (0.5, 5, 50, 1.5, 15, 150 mg/kg) of TiO2-NPs (21 nm) twice a week for 4 weeks.
Each group separated into three subgroups then sacrificed at 4 days, month and 3 months post
intratracheal instillation. The IL-10 and MCP-1 estimation in BALF and histopathological
examination of kidney were done. The results showed serve histological alteration in renal elements
post 4 days post-instillation, which got an increase in a month post instillation as the concentration
of MCP-1 and IL-10 increased in BAlF and in lung tissues homogenate. The histopathological
examination revealed decreasing in glomeruli number in cortex of the kidneys with hemorrhage and
inflammatory cells infiltration at 4 days post-instillation while after a month of instillation, the
changes like swelling, dilatation of Bowman's capsule and degeneration changes in renal tubules
were observed. At 3 months post-instillation, some regeneration in renal elements were observed.
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INTRODUCTION
Titanium dioxide (TiO2), a natural non-silicate mineral oxide, which found in different
forms, is widely used in the pharmaceutical, cosmetic, and paint industries as a coloring material,
However, its special characteristics such as small size, large surface per mass and high reactivity
make it easy to enter into the human body, therefore, poses potential risk to human health and the
environment [1]. TiO2 NPs have two major arrangements of crystal structures, named rutile and
anatase. Both are toxic but the rutile NPs may produce fewer toxic than anatase NPs and these
particles mutually linked with oxidizing mechanisms of an organism, which eligible to produce
reactive oxygen species (ROS) [2]. Fine TiO2-NPs have been counted safe and affectation little
hazard to humans, suggesting that exposed to this material was reasonably harmless. However,
presented data revealed that TiO2-NPs could cause numerous adverse influences on mammalian
cells such as an increase of ROS production and cytokines levels, decrease of cell viability and
proliferation, stimulation apoptosis and genotoxicity [3].
TiO2 NPs formulated products are facilely getting into the human body via diverse paths
with different formats and may object the body metabolism; most of the toxicological studies of
TiO2 NPs in mammalian models have concentrated on the hepatotoxicity through dermal or
inhalation exposure [4]. In 2008, Fabian et al investigated the deposition and removal cycles of
nano-TiO2 in various organs and found that the liver cleaning cycle is the longest (28 days). The
influences of TiO2 in the spleen, lungs, and kidneys are far smaller than in the liver [5]. For
instance, Geyu et al. showed that transbronchial exposure to TiO2 nanoparticles (with a size of 50
nm, and 0.5, 5.0, and 50.0 mg/kg nano-TiO2) can stimulate oxidative stress in the liver and kidney
but has no effect on the liver and kidney function that causes pathological changes [6].
The toxic effects of nano-TiO2 in adult mice have been expert, proposing that higher dose
nano-TiO2 (25 and 80 nm) raising the ratio of alanine aminotransferase to aspartate
aminotransferase, the activity of lactate dehydrogenase and the liver weight, and caused the
hepatocyte necrosis [7]. Recent studies have indicated that TiO2-NPs are toxic on lung, liver,
spleen, kidney, and a gill of animals [8-12].
Moreover, a pathological examination by Chen et al revealed that nano-TiO2 is deposited in
the liver, where it caused liver cell apoptosis, necrosis, and liver fibrosis, and in the kidney, where it
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causes glomerular swelling so nano-TiO2 has been shown to produce toxicity in the liver and
kidneys[13].
Broncho-Alveolar Lavage Fluid (BALF) is usually sampled during bronchoscopy in
individuals with suspected lung cancer, it contains a varied diversity of cellular material such as
macrophages and neutrophils, a large number of proteins produced by epithelial and inflammatory
cells, and tumor cells if present [14]. Proximal biofluids have much reduced the active range of
protein abundances and in some cases are in direct interaction with the site of the disease [15].
In the present study, our aim was to identify altered levels of protein biomarkers in BALF
samples and as the functional integrity of the mammalian kidney is vital to the total body
homeostasis, because the kidney plays a principal role in the excretion of metabolic wastes and in
the regulation of extracellular fluid volume, electrolyte composition, and acid–base balance, so we
investigated the renal histopathological caused by repeated TiO2 NP intake.
MATERIALS AND METHODS
Animals and Treatments
Sixty-three mats rats ( Rattus norvigicus at age 8 weeks) were housed in cages kept in
standard conditions in animals’ room, 25˚C temperature with relative humidity at 60% and a 12
hour light/dark cycle, distilled water and sterilized food for rats were available. Rats were divided
into seven groups (9 rats each). The control group was treated with 0.9% w/w NaCl solution. The
experimental groups treat with 0.5, 5, 50, 1.5, 15, 150 mg/kg of nano-TiO2 (size 21 nm) [11]. All
groups perform to repeated exposure (twice a week, for four consecutive weeks) by 0.1 ml/100 g
(B.W) intratracheal instillation. Animals were sacrificed at 4 days, a month and three months postinstillation;
animals weighted then lungs lavage to collect bronchoalveolar lavage fluid (BALF).
The kidneys were removed, weighted and processed for histopathological study. The coefficients of
tissue to body weight were calculated and it was defined as grams (wet weight of tissue/body
weight).
Collection of Broncho-Alveolar Lavage Fluid (BALF)
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The left lung clamped, and the right lung lavage 3 times with a 3 ml of 0.9% sterile buffer
saline (physiological buffer solution PBS) using a single 3mL Monoject syringe. Lavage fluid
collected in EDTA tubes. Tubes centrifuged at 4°C and 1500 rpm for 3 minutes to isolate alveolar
macrophages from the BALF [16]. BALF supernatant collected for immunological assay.
Preparation of lung homogenates
Lungs sample incised and weighted, immediately freeze in -80˚C for later used. The samples
were hold at 2-8􀀀C. then PBS was added, the samples were homogenized thoroughly by
Homogenizer, and centrifuged at 2000-3000 rpm for 20 minutes, the supernatants collected
carefully, kept and frozen for later used in an immune assay.
Measurements of inflammatory mediators
The levels of interleukin 10 (IL-10), monocyte chemoattract protein -1 (MCP-1), were
measured in the lung homogenate and the BAL fluid supernatants enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer of ELISA Kits (Asiagene, Chania).
Histopathological Examination
Histopathological examinations performed by using standard laboratory procedures.
Kidneys were removed from experimental groups and rinsed thoroughly for 1 mint in normal saline.
Then, the tissue was fixed in a Carnoy's fluid for 60 minutes and transferred to 95% percent or
absolute alcohol for 1 hours. Thereafter, processed to paraffin embedding routine. Sections of 5-7
μm were stained with hematoxylin and eosin stain, collagen stain (Van Geison) and Periodic Acid
Schiffs PAS then examined under light microscope to determine the histopathological changes and
collagen contents [17].
Statistical Analysis:
Statistical analysis of all data was carried out using the ANOVA test with differences less
than 0.5 (p<0.05) considered to be statistically significant. This calculation was carried out
according to the Statistical Package for Social Science (SPSS version 20) and the least significant
difference (L.S.D) at a level less than (0.05) was also used.
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RESULTS
Kidneys coefficients
The results revealed there was a significant decreasing (p<0.05) in kidneys coefficients at 4
days post-instillation in all groups comparing with the control, with no significant differences
between groups. After a month of instillation, the coefficients were increased significantly (p<0.05)
in low doses while the moderate and high doses increased with no significant (p<0.05). After 3
months of instillation, there was decreasing in the coefficients of some groups (0.5, 5, 15&50
mg/kg) and increased significantly (p<0.05) in both doses (1.5& 150 mg/kg) (table: 1)
Table (1): the alteration in kidneys coefficients in treated groups through the experiment.
TiO2 (mg/kg) BW
0 0.5 1.5 5 15 50 150
4 days .555±.012 .3498±.021*a .3615±.037*a .3638±.045*a .4221±.073*a .3676±.09*a .3819±.018*a
Month .5847±0.011 .3957±.026*b .4600±.05*b .3723±.015*a .4526±.032*a .3757±.03*a .4233±.025*a
3 months .6043±.032 .3723±.012*b .5333±.025*c .3606±.029*a .4325±.055*a .3146±.03*b .5000±.05*b
Values represent mean ± SD, (n=3)
* The mean difference is significant (p < 0.05) compared with control
Different letters mean is significant (p<0.05) between periods
Measurements of inflammatory mediators
After 4 days of TiO2 intra-tracheal instillation, the results showed that IL-10 concentration in
tissue homogenate was significantly increased (p<0.05) in lowest doses ( 0.5, 1.5 mg/kg) while
significantly decreased in moderate and high doses (15, 50, 150 mg/kg), and it was significantly
increased (p<0.05) in BALF in all groups compared to the control group, the highest levels of IL-10
were in high doses (50, 150 mg/kg) (Table: 2). After a month and 3 months post-intratracheal
instillation, there was a significant increased (p<0.05) of IL-10 concentration in both homogenate
tissue and BALF of all groups compared with the control group. (Table:2).
The increasing of IL-10 concentration in the homogenate tissue of all groups was significant
during the interval experiment (p<0.05). While IL-10 concentration in BALF of all treated groups
was significantly increased (p<0.05) at 4 days and month post-instillation then it was decreased with
no significantly (p<0.05) at 3 months post-instillation (Fig.1).
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The results showing that the MCP-1 concentration in tissue homogenate after 4 days postintratracheal
instillation was significant (p<0.05) decreased, while the MCP-1 concentration in
BALF was increasing significant (p<0.05) in all groups compared with the control group (Table:1).
After a month and 3 months of TiO2 intra-tracheal instillation, there was a significant (p<0.05)
difference in the mean concentration of MCP-1 in both homogenate tissue and BALF of all groups
compared with the control group as an adjective in the table (2).
There were noticing the concentration of MCP-1 was increased significantly (p<0.05) during
the experiment interval reached the highest after 3 months of instillation in the homogenate tissue of
treated groups compared with the values in 4 days after installation. There was no significant
(p<0.05) increase in the MCP-1 concentration in BALF of treated groups after a month of
instillation compared with their concentration at 4 days of installation, while there was a significant
(p<0.05) difference after 3 months of instillation compared with the concentration at 4 days of
installation. (Fig. 2)
Table (2) IL-10 and MCP-1 concentration in all group after 4 days, 1 month and 3 months of
intratracheal installation TiO2 in BALF and tissue homogenate.
TiO2 NP (mg/kg) body weight
Periods 0 0.5 1.5 5 15 50 150
4days
BALF IL-10 (pg/ml) 87.62±9.32 137.31±11.26a 165.63±27.53a 194.32±10.07a 202.34.±49.56a 255.48±20.81a 339.26±12.51a
*BALF MCP-1
(pg/ml)
109.67±31.82 229.33±32.71a 248.67±25.11a 237.67±53.03a 280.33±28.28a 241.67±28.86a 276.33±20.78a
Homog IL-10 (pg/ml) 136.±33.94 163.24±9.26a 228.61±20.15a 175.45±12.27a 74.05±8.12a 57.09±14.93a 50.08±11.91a
*Homog .MCP-1
(pg/ml)
142.67±35.35 84.67±8.32a 112.67±17.67a 112.67±26.16a 125.33±20.52a 95.33±21.57a 124.33±8.38a
1month
BALF IL-10 (pg/ml) 89.00±5.29 181.98±15.98a 225.38±68.81a 274.88±27.24a 266.49±10.74a 323.89±24.95a 360.89±31.01a
*BALF MCP-1
(pg/ml)
102.33±2.83 233.33±38.19a 216.67±14.43a 284.67±74.19a 237.33±75.43a 242.33±38.30a 235.33±60.01a
Homog IL-10 (pg/ml) 147.18±21.04 250.18±20.66a 257.32±37.14a 271.56±29.77a 246.187±10.67a 264.24±16.19 242.74±20.44a
*Homog .MCP-1
(pg/ml)
157.67±37.1 282.67±61.85a 386.0±85.71a 283.67±83.33a 286.33±86.97a 402.67±25.11a 171.33±15.55a
3months
BALF IL-10 (pg/ml) 87.75±8.07 181.28±32.09a 175.90±33.43a 143.39±12.82a 304±10.11a 265.31±29.73a 308.62±21.85a
*BALF MCP-1
(pg/ml)
107.67±51.08 364.67±50.2a 436.33±27.78a 374.67±9.23a 446.0±14.42a 326.33±1.52a 324.67±67.88a
Homog IL-10 (pg/ml) 153.87±11.84 235.98±11.81a 258.6±35.52a 296.77±34.53a 370.78±22.69a 177.31±40.01a 254.36.±34.3a
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*Homog. MCP-1
(pg/ml)
149.08±17.67 337.33±17.67a 366.33±56.3a 393.67±64.5a 323.33±47.52a 480.33±25.42a 558.33±18.01a
Values represent mean ± SD, (n=3)
a The mean difference is significant (p < 0.05)
Fig. (1) IL-10 concentration in BALF and homogenate lung of all group after 4 days, 1 month and 3 months postintratracheal
installation.
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Fig. (2) MCP-1 concentration in BALF and homogenate lung of all group after 4 days, 1 month and 3 months postintratracheal
installation.
Histopathological Examination
Histopathological examination of kidney at 4 days post intratracheal instillation revealed
that the first three doses (0.5, 1.5 & 5 mg/kg) show more signs of alteration included inflammatory
cells infiltration, hemorrhage in renal tissue, decreased in glomeruli number in cortex. Bleeding in
the glomerulus and epithelial of proximal tubular were necrotic. Dilated and congested blood
vessels and edema appeared in all doses but it was more in high doses (fig.3). When doses (15, 50
& 150 mg/kg) were increased, capsule thickness and giant cells with deposition of collagen fiber
between renal tubules in the cortex (fig. 4). The wall of capillaries in glomeruli showed no
thickening with division into two or three lobules (fig. 5).
After a month of instillation, swelling and dilatation of Bowman's capsule and degeneration changes
in the epithelium of the proximal tubules were observed, which increased as the doses raised. In
addition, bleeding and infiltration of the inflammatory cell were detected in all doses. The edema
sacs became bigger comparing with those in 4 days (fig.3). The collagen fibers were increased
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between renal tubules and around the congested vessels (fig.4). The thickening of the wall of
capillaries in glomeruli increased with eosin materials deposition (fig. 5).
Some regeneration in renal tissue were detected at 3-month post instillation but still
infiltrated with inflammatory cells especially in high doses. The lower doses revealed more
progress in renal tissue remodeling (fig. 3). The collagen fibers were detected in moderate and
high doses (fig.4). The wall of capillaries in glomeruli showed more thickening with division
into two or three lobules in high doses (fig.5)
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Fig. (3) Sections in the kidney from the control and the group treated with (0.5, 1.5, 5, 15, 5
150) mg/kg of TiO2 NP post-instillation, showing decreased in glomeruli number in cortex and
medulla. Bleeding and epithelial of proximal tubular were necrotic post 4 days while swelling and
dilatation of Bowman's capsule and degeneration changes in the epithelium of the proximal
tubules at a month and Some regeneration in renal tissue were detected at 3 months post
installation. H & E stain, 40X
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Fig.(4) Sections in the kidney from control group and the group treated with (0.5, 1.5, 5, 15, 50,
mg/kg of TiO2 NP post-instillation, thicken in capsule and giant cells with deposition of collagen fiber (red
color) between renal tubules in the medulla post 4 days while The collagen fibers were increased between renal
tubules and around the congested vessels at a month. The collagen fibers were detected in moderate and high doses
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Fig.(5) Sections in the kidney from control group and the group treated with (0.5, 1.5, 5, 15, 50, 150) mg/kg
of TiO2 NP post-instillation, showing no thicken in the walls of capillary in glomeruli (C) post 4 days while
the thicken increased at a month and 3 months post-instillation which was deeply pink . PAS stain, 40X.
at 3 months post-installation. Van Geison stain, 40X
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DISCUSSION
TiO2 NPs were used in a variety of applications including dyeing, plastics, papers, inks, food
colorants, toothpaste and cosmetic manufacturing. Our aims were to investigate the
biochemical and histological alterations in the kidney after administration of different doses of
TiO2 NPs. The results at 4 days and a month post instillation revealed that kidneys coefficient
was decreased in all doses due to glomeruli degeneration, which detected through
histopathological examination as well as to the decreasing in glomeruli numbers in both cortex.
While after 3 months there was different in the coefficients due to the increase in edema sacs in
high doses. Meena and Paulraj reported that lower and medium dose nano-TiO2 did not show
any significant differences in the organ to body weight ratios of the liver, kidney, spleen, and
brain of treated animals from control group ,whereas 50mg/kg nano-TiO2, caused a significant
increase in ratio of the liver, kidney, and spleen to bodyweight [18]. There is agreement in
suggesting that nano-TiO2 might damage the organs.
The TiO2 NPs caused increase in IL-10 concentration in the homogenate tissue of all groups
during the interval experiment, while in BALF increased at 4 days and month post-instillation
then it was decreased at 3 months post-instillation, thus indecated the lung inflammation
resulted from TiO2 treatments miaght there were increasing in the experssion levels of
inflammatory factors.
The concentration of MCP-1 was increased during the experiment interval reached the highest
after 3 months of instillation in the homogenate tissue of treated groups compared with the
values in 4 days after installation. However, no significant increase in the MCP-1 concentration
in BALF of treated groups after a month of instillation compared with their concentration at 4
days of installation, while there was a significant difference after 3 months post instillation.
These alteration in MCP-1 secreation due to TiO2 NPs treatment.
It had been reported that nanoparticles (diameter<100 nm) maight cause inflammation easier than
the same mass of fine particles in spite of their chemical properties [19, 20]. Also it has been
demonstrated that nano-TiO2 could promote the expression of several cytokines and chemokines
in the lung of rat and mice, including placenta growth factor (PlGF), MCP-1, IL-1b, and TNF-a
[21,22].
The real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA)
analyses showed that TiO2 NPs can significantly alter the mRNA and protein expression of
several inflammatory pathways, including nuclear factor kappa,light-chain, enhancer of activated
B cells (NF-κB), macrophage migration inhibitory factor (MMIF), TNF-α, interleukin (IL)-6 (IL-
6), IL-1β, cross-reaction protein, IL-4, and IL-10 [23]. Intraperitoneal injection for 14 days
indicated that the titanium content increased in mouse livers, resulting in liver cell damage,
mitochondrial swelling, and other pathological changes, while the expression levels of
inflammatory factors (NF-κB, MIF, IL-6, IL-1β, CRP, TNF-α, etc) were altered, indicating that
nano-TiO2 causes liver inflammation that results in liver injury [10]. The expression of the
inflammation-associated molecule, monocyte chemoattractant protein-1 (MCP-1) and
macrophage marker-CD11b was decreased in glomeruli in mice [24].
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the kidney. Moreover, particles of 25 nm TiO2 can significantly increase the urea level of serum
compared with the control group [7]. Vasantharaja et al. suggested that oral administration of TiO2
NPs aggregated in the liver and kidney [4]. After inhalation of nano-TiO2, this nanoparticle
accumulates in the kidneys and causes renal fibrosis via oxidative stress [25]. The effects of TiO2 on
Kidney included infiltration of inflammatory cells, fatty degeneration, and apoptosis; degeneration
of superficial adipocytes; apoptosis of renal tubules [25]. The lymph nodes was described as the
first target of NPs after lung translocation [27,28]. However, Pujalté, et al. study, like other published
studies, failed to distinguish between direct transmit of NPs into the interstitium and then the blood,
and transmit through lymphatic circulation to blood, nevertheless, with the exposure conditions
used, the TiO2 NPs translocation average to the lymphatic system and the blood circulation was
found to be low compared to total amounts of NPs hooked up in the lungs through time [29].
CONCLUSION
The high doses (15, 50, 150) mg/kg of TiO2 NPs had more effects on immune response which
remain for 3 months post-instillation through its stimulating to secretion of IL-10, and MCP-1
where it raised in BALF and decrease in tissue homogenate. After post instillation, the low doses
caused more alteration in the renal histological structures (included decreasing in glomeruli number,
necrotic proximal convoluted tubules, division the glomeruli into more than one lobules and
The results of histopathological showed decreasing in glomeruli number in cortex.
Bleeding, epithelial of proximal tubular were necrotic post 4 days while swelling, dilatation of
Bowman's capsule and degeneration changes in the epithelium of the proximal tubules at a
month while some regeneration in renal tissue were detected at 3 months post-installation, thus
because of the TiO2 NPs accumulate in such organs which practically eliminate the toxic
compounds. Accumulation of these substances caused abnormal pathological changes in the
tissues of the lung and liver [11, 12]. Wang et al. consider that nanoparticles of TiO2 have been
deposited in the cells of kidney and caused the pathological alterations and nephron-like toxicity
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collagen fibers deposition) which raised after month post-instillation in all doses. There were some
regeneration in renal tissue after 3 months post-instillation.
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