2. CHAPTER 2 MATERIALS AND METHODS
2.3 Protein electrophoresis, detection and confirmed by MALDI-TOF/TOF mass
For sodium dodecylacrylamide gel electrophoresis (SDS-PAGE), the protein
sample was mixed with 5 x sample treatment buffer (125 mM Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 5%-merceptoethanol, and 0.05% bromophenol blue), and heated
at 100 °C for 10 min. Electrophoresis was performed according to the manufacturer’s
instructions. After electrophoresis, the gel was soaked in Coomassie Blue R 250
staining solution for 30 min, then the gel was destained with the destaining solution I
13
(40% methanol, 7% acetic acid) and II (5% methanol, 7% acetic acid) until the stained
band was distinct against a clear background. The protein identities of SDS-PAGE
bands corresponding to Gh-rTDH were confirmed by MALDI-TOF/TOF mass
spectrometry.
2.4 Analyzed the in vitro hepatotoxicity of Gh-rTDH
2.41 Cytoviability and morphological examination of Gh-rTDH treated human liver
cell and FL83B cells
FL83B (BCRC 60325) and primary human non-cancer cell (kindly provided by
the Liver Transplantation Center of one medical center in central Taiwan; IRB number:
120305) were cultured for use in these studies. Following attachment, the cells were
treated with Gh-rTDH at a concentration of 1 μg/ml for 24 hours at 37 °C; the treating
dose was determined according to the initial results of the IC50 determination (1 μg/ml,
obtained from MTT assay). Images of the experimental group, cellular morphology
were recorded microscopically at 4 time points (before Gh-rTDH exposure and after
exposure to Gh-rTDH for 8, 16, and 24 hours). In addition, cells treated with PBS
(mixed with culture medium) were served as control group, they were also observed
at the same time points with the experimental group. All experimental or control
groups under the same conditions. Hygromycin was not used in this study.
14
2.42 Cytoviability assay
Cytoviability of human liver cell and FL83B cells were measured by the MTT
assay using 4 treatment durations (12, 16, 24 and 48 hours). In the MTT assay, cells
were treated with PBS as control groups and treated with Gh-rTDH at different
concentrations (10to 10-8 μg/ml mixed with culture medium and administered in a total volume of 250 μl). For control group, the same concentration of vehicle was
added to the culture medium. After culture for different treating durations (12hours,
16hours, 24hours and 48hours), cells were incubated with MTT for another 4 hours at
37°C. Overall, the medium was removed and DMSO was added into each well. The
absorbance of the samples was measured at 570 nm using a microtiter plate reader. All
experiments were performed independently for five times
2.43 Confocal microscopy
Confocal microscopy was used to investigate the locations where Gh-rTDH
invaded in liver cells. Gh-rTDH was conjugated with fluorescein isothiocyanat (FITC)
(emission 488nm, green) as Gh-rTDH-FITC and the reactions were performed using
the FluoReporter FITC Protein Labeling Kit (molecular probes) according to the
manufacturer’s protocol. FL83B cells were seeded in 8-well chamber slide (1×104
15
cells/well) and incubated in the culture medium to attach. After cells were attached,
they were treated with 10 μg/ml of Gh-rTDH-FITC mixed with culture medium for 20
and 40 min in darkness. Subsequently, the cells were washed 3 times using PBS
(SIGMA) buffer and they were also stained with propidium iodide (PI) (SIGMA)
(emission 650 nm, red) with working solution 5mg/ml in PBS for 5 min in darkness.
Finally, the cells were washed 3 times using PBS buffer and observed at 26 °C by
confocal microscopy (Olympus FV300).
2.44 TUNEL assay
TUNEL assay was performed for analyzing the reason of cell death. FL83B cells were respectively administrated with 1 μg/ml of Gh-rTDH for 24 hours and PBS
(control group) and the result of TUNEL assay according to the manufacturer’s
protocol (ApoAlert® DNA Fragmentation Assay Kit) and were observed by confocal
microscopy (Olympus FV300).
2.5 Analyzed the in vivo hepatotoxicity of Gh-rTDH 2.51 BALB/c served as an in vivo model
A total of 114 female mice aged 6 weeks were obtained from the National
Laboratory Animal Center of Taiwan and were used to analyze in vivo hepatotoxicity.
16
All mice were fed normal diets. This study was carried out in strict accordance with
the recommendations in the Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health. The protocol was approved by the Committee on the
Ethics of Animal Experiments of the National Chiao Tung University (Permit
Number: 01001008). All surgery was performed under sodium pentobarbital
anesthesia, and all efforts were made to minimize suffering.
2.52 Withdraw blood for analyzing the liver functions (n=25)
Twenty-five mice were divided into 5 groups (n = 5 for each group). One group
served as a control group and was administered PBS; the other 4 groups were
administered different dosages of Gh-rTDH (0.1, 1, 10, and 100 μg) as a single
treatment. The dosage that might initiate organ injury in animals has never been
reported (information on natural infection in humans is also lacking). Therefore, the
treatment dosages were carefully determined and modified according to the initial
results of the IC50 determination (1 μg/ml, obtained from the MTT assay described above). All mice were treated using the same volume (200 μl), the same treatment
time (10:00 am) and via gastric tubes without volume loss (i.e., vomiting). One
hundred microliters of whole blood was withdrawn from the orbital vascular plexus of
each mouse using a capillary tube and no analgesics. There were 8 experimental
17
sampling times: before treatment with PBS or Gh-rTDH and 4, 8, 16, 32, 64, 128 and
256 hours after treatment with PBS or Gh-rTDH. The blood samples were analyzed
for the continuation of liver function as assessed by glutamic-oxaloacetic
transaminase (GOT), glutamic-pyruvic transaminase (GPT), total/direct/indirect
bilirubin, albumin and globulin)(Reagents Beckman Coulter®). One-way ANOVA
analysis was used to analyze the significant differences between each treatments/time
point. All analyses were performed with the SPSS statistical package for Windows
(Version 15.0, SPSS Inc., Chicago, IL).
2.53 Withdraw blood for analyzing the cardiotoxicity and nephrotoxicity (n=20)
Twenty mice were divided into 4 groups (each n=5). One of the 4 groups was
served as control group which were fed with PBS and the other 3 groups were
respectively fed with Gh-rTDH in dosages of 1 μg, 10 μg and 100 μg in single
administration via gastric tubes (each mouse was fed at AM10:00 with total volumes
of 200 μl). Each mouse was also respectively withdrawn 100 μl of whole blood at the
5 different time points: (1) before feeding with PBS or Gh-rTDH; (2) after feeding
with PBS or Gh-rTDH for 4, 16, 64, and 256 hours. Their blood samples were
analyzed for nephrotoxicity by detecting the level of creatinine (Assay kit: Creatinine
Reagent, Beckman Coulter®; Supply: Beckman Coulter Synchron CX7 Analyzer) and
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for cardiotoxicity by detecting the levels of CK-MB (Assay kit: CK-MB Reagent
Pack, Beckman Coulter®; Supply: Beckman Coulter Synchron CX7 Analyzer) and
Troponin I (Assay kit: ADVIA Centaur TnI-Ultra Readypack®; Supply: Bayer ADVIA
Centaur). Above all, blood samples were diluted appropriately for enough volume to
be detected by analyzers and were operated according to the manufacturer’s protocol.
One-way ANOVA analysis was also used to analyze the significant differences
between each treatments/time point.
2.54 Liver biopsy (n=9)
Nine mice were divided into 3 groups, which were treated with PBS, 10 μg of
Gh-rTDH or 100 μg of Gh-rTDH (n = 3 in each group) in a single administration via a
gastric tube. All mice had their livers biopsied after 8 hours of treatment. Samples
were prepared with H&E staining from tissue harvested at the time of animal
sacrifice.
2.55 PET/CT scan (n=60)
In this study, the 18F-FDG (2-fluoro-2-deoxy-D-glucose) PET /CT scan was
used to take images in detection the liver cells metabolism in living animals after
exposure of Gh-rTDH and their trends were recorded (GE Medical System, Discovery
19
ST). 18F-FDG PET/CT imaging provides precise fusion of molecular PET images
with high-quality anatomical CT images. Technical parameters used for CT portion of
PET/CT are designed as follow: CT scan type with helical full of 0.5 second, a
detector row configuration of 16x1.25mm, an interval space of 2.5 mm, the slice
thickness of 1.2 mm, pitchof 1.75:1 (high quality mode), a speed of 17.5mm per
rotation, scan FOV of large, voltage of 120 kVp and current of 200 mA. Technical
parameters used for PET portion of 18F-FDG PET/CT are designed as follow 10 min
in each bed, the FOV chosen for imaging reconstruction is 20 cm and PET resolution
is 4.5 mm FWHM. The reconstructive parameters are type 3D iteration.
Sixty mice were divided into 4 major groups and each group (n=15) was respectively
fed with PBS, 1 μg, 10 μg and 100 μg of Gh-rTDH in single administration via gastric
tubes. Among each group, mice were further grouped to receive 18F-FDG PET/CT
scan in different time points including the 8th (n=5), 72th (n=5) and 168th (n=5) hours
after feeding with Gh-rTDH. In the study, 0.07mCi 18F-FDG for each mouse was
given by tail vein injection before taking the image (Figure 7A). After injection the
18F-FDG, images taking were performed one hour later with appropriate general
anesthesia (Isoflurane) (Figure 7B-D). In our study, each mouse did not be proposed
to receive 18F-FDG PET/CT scan in every time points to follow up because of
recurrent general anesthesia in short time might cause severe hepatotoxicity and could
20
influence the results of this study. In the 18F-FDG PET/CT images, the 18F-FDG
uptake value was calculated using region of interest (ROI). In each mouse, the ROIs
of liver and muscle were recorded for semi-quantification which was proposed to be
the ratios of liver/muscle 18F-FDG uptake level.
Figure 7 18F-FDG and Isoflurane were treated to each mouse before scan started. (A)
18F-FDG for each mouse was given by tail vein injection before taking the images. (B)
21
Isoflurane was used to perform general anesthesia. (C) Mice were respectively lay
down on the box and (D) received 18F-FDG PET/CT scan.
2.6 Infection models –In vivo hepatotoxicity of the G. hollisae strain, Escherichia coli containing the recombinant Gh-tdh gene (E. coli-TOPO-tdh), and the E.
coli-TOPO strain in BALB/c mice (n=126).
An animal infection model was set up to demonstrate the hepatotoxicity of
bacterial infection. The G. hollisae strain (wild type), E. coli-TOPO-tdh, and E.
coli-TOPO strains were cultured. Seventy-five mice were divided into three major
groups (n=25 for each group) and infected with bacteria via oral administration. Two
groups were infected with G. hollisae and E. coli-TOPO-tdh to demonstrate their
hepatotoxicity; the third group was infected with E. coli-TOPO to serve as a control
group. For each major group, five subgroups were established (n=5 for each group)
according to their treatment dosage (107, 108, 109, 1010 and 1011 organisms/ml and
treated with the same volumes. One hundred microliters of whole blood was
withdrawn at 8 different time points: before treatment with bacteria and 4, 8, 16, 32,
64, 128 and 256 hours after treatment with bacteria. Blood samples were analyzed for
continued liver function (GOT, GPT, total bilirubin, albumin and globulin). In
addition, 6 mice were treated with 1011 organisms/ml of G. hollisae, E. coli-TOPO-tdh,
22
and E. coli-TOPO (n=2 for each group). For these animals, liver biopsies and H&E
staining (200X) were performed 8 hours after bacterial treatment. Finally, 54 mice
were treated with G. hollisae, E. coli-TOPO-tdh and E. coli-TOPO (n=18 for each
group) with a single administration. Among each group, mice were sub-grouped for
treatment with bacteria at the concentrations of 107, 109 and 1011 organisms/ml (n=6
for each group). In each concentration group, mice received a PET/CT scan at 8, 72
and 168 hours (n=2 for each group) after bacterial treatment.
2.7 Analyzed the in vivo and in vitro hepatotoxicity of fiber from Gh-rTDH (n=34)
In this study, fiber form of Gh-rTDH was prepared by heat treatment at 60 °C,
and the aggregates were collected by centrifugation. The method of preparing fiber
form of Gh-rTDH was according to a previously described method.(16) The
productions of fiber form of Gh-rTDH were confirmed by testing their hemolytic
ability. FL83B cells were treating with fiber form of Gh-rTDH and morphological
examination and cytoviability assay were performed (procedures and conditions were
uniform with previous description in the method section of 2.4). Moreover, mice
(n=25) were also fed with fiber form of Gh-rTDH and their liver functions including
the levels of GOT, GPT, total bilirubin, albumin and globulin were recorded. Liver
23
biopsy and pathological images were taken in mice (n=8) for further analyzing the
hepatotoxicity of mice (procedures and conditions were uniform with previous
description in the method section of 2.5).
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Chapter 3 Results
3.1 Identification of the Gh-rTDH purified from G. hollisae
Electrophoresis of the homogeneous protein revealed a molecular mass of ~ 22
kDa as determined by the SDS-PAGE (Figure 8). Moreover, we found that tandem
mass spectrum of the doubly charged tryptic peptide at m/z 1024.543 from
SDS-PAGE of Gh-rTDH and a unique hit matching the 35VSDFWTNR42 of Gh-rTDH
peptide sequence was identified from the mass differences in the y-fragment ion series
of MALDI TOF/TOF spectrum (Figure 9).
25
Figure 8 Purification and characterization of the Gh-rTDH protein. (A) Coomassie blue-stained SDS-PAGE of Gh-rTDH protein. Marker proteins (M): phosphorylase b
(97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa),
trypsin inhibitor (20 kDa), α-lactoalbumin (14 kDa); lane 1: cell crude extract of
BL21(DE3) pLysS strain containing pCR2.1-TOPO plasmid alone; lane 2: crude
protein expressed from BL21(DE3) pLysS strain containing pCR2.1-TOPO-Gh-tdh
gene; lane 3 and 4: Phenyl Sepharose 6 Fast Flow purified protein showed a
homogenous protein with a molecular mass of ~ 22 kDa.
26
Figure 9 Tandem mass spectrum of the doubly charged tryptic peptide at m/z 1024.543 from SDS-PAGE of Gh-rTDH. A unique hit matching the 35VSDFWTNR42
of Gh-rTDH peptide sequence was identified from the mass differences in the
y-fragment ion series of MALDI TOF/TOF spectrum.
3.2 Gh-rTDH caused in vitro liver cell damage
The morphology of liver cells was obviously changed after administrating with
1 μg/ml Gh-rTDH for 24 hours at 37 °C. The morphological changes included cell
detachment, and loss of cell cytoplasm with cell shrinkage (Figure 10A-D). The MTT
assay also revealed that the cytoviability of liver cells decreased in proportion to the
concentrations of Gh-rTDH in different treating durations. Moreover, we noted that
the Gh-rTDH damaged the liver cells in vitro when the concentration of Gh-rTDH
crossed 10-6 μg/ml (Figure 11). Moreover, in this study, primary human hepatocytes
(non-cancer liver cells) were used to demonstrate the toxicity of Gh-TDH via MTT
assay. These primary human hepatocytes were kindly provided by the liver
transplantation center of a medical center in central Taiwan under IRB permission
(IRB number: 120305). In this MTT assay, the Gh-TDH still caused obvious
hepatotoxicity in primary human hepatocytes (Figure 12).
27
Figure 10 The morphology of liver cells (FL83B) was clearly changed after the administration of 1 μg/ml Gh-rTDH for 24 hours at 37 °C. The morphological
changes included cell detachment and a loss of cell cytoplasm with cell shrinkage;
they were the same cells recorded in different time points. (A) The liver cells before
exposure and (B) after exposure to the Gh-rTDH protein for 8 hours, (C) for 16 hours
and (D) for 24 hours.
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Figure 11 The MTT assay of mouse liver cells. The MTT assay revealed that the cytoviability of mouse liver cells decreased in proportion to the concentration of
Gh-rTDH over different treatment durations. Moreover, we noted that Gh-rTDH
damaged liver cells in vitro when the concentration of Gh-rTDH exceeded 10-6 μg/ml.
29
Figure 12 The MTT assay of human liver cells. The MTT assay revealed that the cytoviability of primary human hepatocytes (non-cancer liver cells) decreased in
proportion to the concentration of Gh-rTDH over different treatment durations.
Moreover, we noted that Gh-rTDH damaged liver cells in vitro when the
concentration of Gh-rTDH exceeded 10-8μg/ml.
3.21 Gh-rTDH-FITC bound the margin of liver cells and invaded their nucleuses
Gh-rTDH-FITC was used to demonstrate the locations where the protein
invaded. The confocal microscopy with FITC filter revealed that Gh-rTDH-FITC
bound around the margin of liver cells after administrating with 10 μg/ml Gh-rTDH-FITC for 20 min at 26 °C (Figure 13 A-C). Moreover, Gh-rTDH-FITC
further located in the nucleus of liver cell after treating with Gh-rTDH-FITC for 40
30
min at 26 °C and PI was also stained for confirming the location of nucleus (Figure 13
D-F).
Figure 13 Subcellular localization of Gh-rTDH. The liver cells respectively administrated with 10 μg/ml Gh-rTDH-FITC for 20 (A-C) and for 40 (D-F) min at 26
°C and were observed by confocal microscopy. (A) The liver cells were observed
without FICT filter (B) with FITC filter (C) merge A and C confirmed that
Gh-rTDH-FITC (green) bound around the margin of liver cells. (D) The nucleus of
31
liver cell was invaded by Gh-rTDH-FITC (green) (E) nucleus stained with PI (red) (F)
merge of D and E confirmed that Gh-rTDH-FITC was located in the nucleus of liver
cell.
3.22 Gh-rTDH caused liver cells death by cell apoptosis
After treating liver cells with 1 μg/ml of Gh-rTDH for 24 hours, TUNEL assay
was performed and positive findings were noted. DNA fragmentations could be noted
in the cytoplasm of cells (Figure 14 A-D). In the control group, TUNEL assay was
negative finding (Figure 15).
32
Figure 14 The liver cells were administrated with 1 μg/ml of Gh-rTDH for 24 hours and performed with TUNEL assay were observed by confocal microscopy. (A)
TUNEL positive, (B) stained with PI, (C) merge of A and B, (D) merge of C and
image without filter. Arrows: DNA fragmentations
33
Figure 15 In the control group, the liver cells were administrated with PBS for 24 hours and performed with TUNEL assay was observed by confocal microscopy. (A)
TUNEL negative, (B) stained with PI, (C) without filter, (D) merge of C and D
34
3.3 Liver damages in vivo were induced by Gh-rTDH
The levels of GOT and GPT were not elevated in the control group after the
administration of a single dose of PBS. However, the mean GOT and GPT levels were clearly elevated in the group that was treated with 0.1 μg Gh-rTDH, and the highest
levels were observed 8 hr after toxin administration. Similar findings were observed
in other treatment groups. Higher doses of Gh-rTDH were clearly associated with
more severe mouse liver injury (Figures 16).
35
Figure 16 Liver function evaluation after a single administration of Gh-rTDH. The levels of liver function were abnormal after a single administration of Gh-rTDH.
Six-week-old female BALB\c were treated with four different dosages of Gh-rTDH
36
(0.1, 1, 10, and 100 μg), and the control group was treated with PBS (n = 5 for each
group). Acute liver injury was demonstrated by elevating the levels of (A) GPT and
(B) GOT; the highest levels could be found at the 8th hr after feeding in both. (C)
Hyperbilirubinemia and (D) hypoalbuminemia also occurred in the mice that were
treated with Gh-rTDH. The hyperbilirubinemia was the most severe at the 8th hr, and
hypoalbuminemia was noted after 32 hr of treatment with Gh-rTDH. (E) Globulin
levels were gradually increased after exposure to Gh-rTDH. *A p-value < 0.05 was
considered statistically significant.
3.31 Acute hemolytic status in vivo was caused by Gh-rTDH
In addition, the total bilirubin level did not change in the control group (Figure
16 C) the distributions of bilirubin were similar in different time points after exposure
with PBS (Figure 17 A). However, in the groups fed with Gh-rTDH, their total
bilirubin levels were obviously elevated and the higher dosage of Gh-rTDH made the
levels of total bilirubin higher (Figure 17 C). Moreover, the proportions of indirect
from bilirubin were much higher than the direct from of bilirubin within 8 hours after
exposure with Gh-rTDH in the dosages of 1 μg and 100 μg (Figure 17 B and C).
According to our findings, the acute hemolytic status in vivo could be induced by
feeding with Gh-rTDH.
37
Figure 17 Gh-rTDH induces an acute hemolytic status. The distribution of direct and indirect bilirubin in mice that were fed with (A) PBS (control), (B) 1 μg of Gh-rTDH, or (C) 100 μg of Gh-rTDH. The percentages of indirect form of bilirubin were similar
in different time points after exposure to PBS. In mice fed with 1 μg and 100 μg of
Gh-rTDH, the percentages of indirect form of bilirubin both obviously increased in
the 4th hour and gradually subsided. This result indicated that the Gh-rTDH caused
acute hemolytic status in vivo and the severity associated with the feeding dosages of
toxin.
38
3.32 Gh-rTDH damaged the functions of albumin synthesis in liver and triggered
immune system
The albumin levels began to decrease after feeding with Gh-rTDH for 32 hours
and were in proportion to the feeding dosages. Moreover, in the groups fed with
Gh-rTDH, their albumin levels progressively decreased and which did not recover
even in the 256th hour (Figure 16 D). These results indicated that in the mice fed with
even in the 256th hour (Figure 16 D). These results indicated that in the mice fed with