1. CHAPTER 1 INTRODUCTION
1.4 Research summary…
Previous studies emphasized the toxin effect of TDH mainly located in the
intestine of gastrointestinal tract.(15) However; the relationships between TDH and
liver had not been reported or analyzed. TDH might not only be absorbed by intestine
but also probably caused secondary injury to the liver via venous return of portal
system (Figure 4). In this study, we aim to analyze the hepatotoxicity of TDH and G.
hollisae (toxin and infection model) by in vitro (Figure 5) and in vitro (Figure 6)
analyses and provide an evidence to report the acute injury and recover stages of liver
in living animals by 18F-FDG PET (positron emission tomography)/CT (computer
tomography) scan.
Figure 4 TDH might not only be absorbed by intestine but also probably caused secondary injury to the liver via venous return of portal system.
Seafood G.
hollisae
TDH Intestine Portal
vein Liver
9
Figure 5 The parameters in analyzing the in vitro hepatotoxicity of TDH from G.
hollisae. Mouse and human liver cell served as an in vitro model.
Figure 6 The parameters in analyzing the in vivo hepatotoxicity of TDH from G.
hollisae and G. hollisae. BALB/c served as an in vivo model.
Morphological
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Chapter 2 Materials and Methods
2.1 Bacterial strains and materials
The G. hollisae strain ATCC 33564 was obtained in a freeze-dried form from
the Culture Collection and Research Center (Hsin-Chu, Taiwan). Phenyl Sepharose 6
Fast Flow and protein molecular weight standards were purchased from GE
Healthcare (Piscataway, NJ). The protein assay kits were obtained from Bio-Rad
(Hercules, CA). Protein purification chemicals were obtained from Calbiochem (La
Jolla, CA).
2.2 Molecular cloning, protein expression and purification, and characterization of G. hollisae recombinant thermostable direct hemolysin (Gh-rTDH)
The G. hollisae tdh gene was obtained via PCR using G. hollisae genomic DNA
as the template and two primers, YKW-hol-TDH-N1
(5’-ATGAAATACAGACATCT-3’) and YKW-hol-TDH-C1
(5’-TTATTGTTGAGATTCAC-3’). PCR reaction was carried out under the following
conditions: denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at
94 °C for 15 s, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min
followed by a final extension at 72 °C for 10 min. The amplified DNA fragment was
cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) vector in order to construct the
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recombinant plasmid pCR2.1®-TOPO-Gh-tdh. The recombinant plasmid was
sequenced using an ABI PRISM® 3100 Genetic analyzer according to the
manufacturer’s protocol (Applied Biosystems, Foster City, CA).
The pCR2.1-TOPO-Gh-tdh plasmid harboring the tdh gene was transformed
into Escherichia coli BL21(DE3)(pLysS) cells (Promega, Madison, WI) for
recombinant protein production and purification. In parallel, the pCR2.1-TOPO
plasmid was used as a negative control. Colonies were inoculated into Luria-Bertani
broth supplemented 50 g/mL kanamycin and grown for 16 hours at 37 °C. The cells
were harvested by centrifugation at 6,000 x g for 30 min, and then resuspended in 40
mL of 20 mM Tris-HCl (pH 7) buffer. The mixture was sonicated, and the cell debris
was removed by centrifugation at 12,000 x g for 30 min at 4 °C. Purification method
was according to a previously described method.25 Overall, the supernatant containing
produced Gh-rTDH protein was loaded on a Phenyl-Sepharose 6 Fast Flow column
pre-equilibrated with 20 mM Tris-HCl (pH 7) and eluted with a linear 0 to 50%
ethylene glycol gradient. Fractions exhibiting hemolysis were pooled, dialyzed, and
added with NaCl to a 200 mM concentration. The active sample was applied to a
Phenyl-Sepharose 6 Fast Flow column with 20 mM Tris-HCl (pH 7) and then eluted
with 4 void volumes of a step gradient consisting of 200, 100, 50 mM NaCl and 20
mM Tris-HCl (pH 7) buffers. Finally, the protein (Gh-rTDH) was eluted by a 20 mM
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Tris-HCl buffer and the Gh-rTDH was dialyzed against 0.1M PBS buffer (Na2HPO4,
NaH2PO4, NaCl, pH 7.1) at 4°C overnight for cell and animal experiments. The
molecular cloning, protein expression, and purification of Gh-rTDH were performed
according to previous reports (17, 24). The effect of endotoxin has been excluded
before the experiment started. In this study, the endotoxin contamination had been
excluded during protein preparation by the method of anion-exchange
chromatography using diethylaminoethane (DEAE) chromatographic matrices.(25, 26)
Other prevention strategies including staff education (the use of aseptic technique,
understanding the nature of contamination, and good housekeeping), and sterility tests
were routinely performed before the study started.
2.3 Protein electrophoresis, detection and confirmed by MALDI-TOF/TOF mass spectrometry
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
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(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.
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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
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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
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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
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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)
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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
without FICT filter (B) with FITC filter (C) merge A and C confirmed that