霍氏格里蒙菌熱穩定性溶血素造成活體內外肝臟毒性的探討

國立交通大學

生物科技學系

博士論文

霍氏格里蒙菌熱穩定性溶血素造成活體

內外肝臟毒性的探討

Thermostable direct hemolysin from

Grimontia hollisae causes in vitro and in vivo

hepatotoxicity

研究生：林晏任

Student: Yan-Ren Lin

指導教授：吳東昆 博士

Advisor: Prof. Tung-Kung Wu Ph.D

霍氏格里蒙菌熱穩定性溶血素造成活體

內外肝臟毒性的探討

Thermostable direct hemolysin from Grimontia

hollisae causes in vitro and in vivo hepatotoxicity

研究生：林晏任 Student: Yan-Ren Lin

指導教授：吳東昆 博士 Advisor: Prof. Tung-Kung Wu Ph.D

國立交通大學

生物科技學系

博士論文

A Dissertation

Submitted to Institute of Biological Science and Technology,

National Chiao Tung University,

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Biological Science and Technology Hsinchu,

Taiwan, Republic of China

Feb, 2013

i

摘要

究中，我們將進行熱穩定性溶血素造成活體內外肝臟毒性(in vitro and in vivo

hepatotoxicity)的探討。我們使用純化的 G. hollisae recombinant thermostable direct

hemolysin (Gh-rTDH) 來進行 肝 毒性 的分析 。在活 體外 培養老鼠 肝 臟細胞株

(FL83B cell line)和人類正常肝臟細胞(primary human liver cell)來測試此毒素後發

現，此毒素對老鼠和人類的肝臟細胞均造成劇烈的傷害。在接觸此毒素後，肝臟

細胞出現細胞質流失和皺縮等(loss of cell cytoplasm with cell shrinkage)型態上的

改變，並且最終造成細胞死亡。細胞毒性測試(MTT assay)的結果顯示當毒素濃

度超過 10-6_{μg/ml 則開始顯著造成肝細胞死亡，作用時間越長或毒素的濃度越高，}

則毒殺肝細胞的作用就更為明顯(dose and time dependent)。Gh-rTDH 在結合異硫

氰酸螢光素(fluorescein isothiocyanate-conjugated)之後被用來定位細胞受侵犯的

部位，我們發現此毒素起初會圍繞肝細胞，最後將進入細胞內並且侵犯細胞核造

成細胞死亡。另外，在活體內的實驗中，我們讓若干數量的小鼠(BALB/c)口服單

次且不同劑量的熱穩定性溶血素或霍氏格里蒙菌，並且進行抽血分析以評估其肝

ii

glutamic-pyruvic transaminase) 、 膽 紅 素 (bilirubin) 、 白 蛋 白 (albumin) 、 球 蛋 白

(globulin)在餵食毒素或細菌後均發生顯著異常，肝臟酵素在餵食後第八小時上升

至最高峰。病理切片則證實無論是餵食毒素或直接餵食細菌，在肝臟的門脈周圍

區域(periportal area)皆會遭受明顯的破壞，毒素或細菌的濃度越高，破壞越大。

除此之外，此毒素對於心臟、腎臟並沒有造成顯著的傷害。最後，我們使用正子

照影18

F-FDG PET (positron emission tomography)/CT (computer tomography) scan

來活體評估老鼠在餵食毒素或細菌前後之肝臟代謝功能及復原情形。結果發現老

鼠在餵食毒素或細菌後，其代謝功能有顯著下降，並且在七天後可逐漸回復其功

能。總結來說，本研究首次證實霍氏格里蒙菌之熱穩定性溶血素的確具有活體內

外之肝臟毒性，肝臟的門脈周圍區域是此毒素主要的侵犯部位，肝臟代謝功能可

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Abstract

G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G.

hollisae. This toxin has been reported to be absorbed in the intestines in humans. In

addition to being absorbed by the intestine, TDH may also cause secondary injury to

the liver via effects on the venous return of the portal system. However, a relationship

between the TDH and the liver has not been reported or analyzed. In this study, we

analyzed the hepatotoxicity of TDH in vivo and in vitro to provide insights into the

acute injury and recovery stages of THD-induced hepatotoxicity in living animals.

Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and

mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. In this study,

G. hollisae recombinant thermostable direct hemolysin (Gh-rTDH) was purified for

further analysis. Morphological examination and cytotoxicity assays using liver cells

were also performed. The morphological changes included cell detachment and a loss

of cell cytoplasm with cell shrinkage. The MTT assay revealed that the cytoviability

of liver cells decreased in proportion to the concentration of Gh-rTDH over different

treatment durations (dose and time dependent.). Moreover, we noted that Gh-rTDH

damaged liver cells in vitro when the concentration of Gh-rTDH exceeded 10-6μg/ml.

Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of

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located around the cellular margins and subsequently entered the nucleus. Mice were

subjected to liver function measurements and liver biopsies following toxin treatment

and wild-type bacterial infection. Liver function measurements and liver biopsies of

the mice following treatment with toxin or infection with wild-type Grimontia

hollisae showed elevated levels of transaminases and damage to the periportal area,

respectively. PET (positron emission tomography)/CT (computed tomography)

images were taken to assess liver metabolism during acute injury and recovery. The

PET/CT images revealed that the reconstruction of the liver continued for at least one

week after exposure to a single dose of the toxin or bacterial infection. In conclusion,

the hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in

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誌謝 (Acknowledgement)

從一位急診臨床醫師跨入生物科技這個有點熟悉但又相當

陌生的領域當中。起初雖因反覆實驗失敗而充滿徬徨，但過

程中我仍學習到許多基礎科學的知識、實驗的方法，最重要

的是嚴謹的實驗態度。首先要感謝我的指導教授吳東昆老師，

體諒我在職的身分，並在此基礎與臨床結合的題目上，給予

解惑及最大的協助鼓勵。再來要感謝生科所的諸位學長姐、

學弟妹的幫忙，尤其是裕國、文亮、聖慈、宜芳的全心的付

出與大力的支持，如果沒有你們幫忙，整個過程是無法獨自

完成的。感謝光田醫院核醫團隊對於動物影像實驗的幫忙與

指導。最後，感謝彰化基督教醫院、交大應化諸位長官、師

長的支持與照顧，與一路相伴的家人，你們都是我堅強的後

盾，謝謝大家。

vi

TABLE OF CONTENT

1.1 Vibrio species caused diseases throughout the world………...………1

1.2 Grimontia hollisae cause diseases in human………3

1.3 Thermostable direct hemolysin was a violence factor……….….…...….4

1.4 Research summary…...8

2. CHAPTER 2 MATERIALS AND METHODS………..…...10

2.1 Bacterial strains and materials……….…...…10

2.2 Molecular cloning, protein expression and purification, and characterization of G. hollisae recombinant thermostable direct hemolysin (Gh-rTDH)…...10

2.3 Protein electrophoresis, detection and confirmed by MALDI-TOF/TOF mass spectrometry………...….12

2.4 Analyzed the in vitro hepatotoxicity of Gh-rTDH………..13

2.4.1 Cytoviability and morphological examination of Gh-rTDH treated human liver cell and FL83B cells………..………..13

2.4.2 Cytoviability assay………..……….…...…14

2.4.3 Confocal microscopy……….…..…….14

2.4.4 TUNEL assay ………...………..….……15

2.5 Analyzed the in vivo hepatotoxicity of Gh-rTDH………15

2.5.1 BALB/c served as an in vivo model………...…..15

2.5.2 Withdraw blood for analyzing the liver functions………..…..16

2.5.3 Withdraw blood for analyzing the cardiotoxicity and nephrotoxicity……..17

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2.5.5 18F-FDG PET/CT scan………..…..…...…..18 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………...21

2.7 Analyzed the in vivo and in vitro hepatotoxicity of fiber from Gh-rTDH…...…22 3. CHAPTER 3 RESULTS……….…24

3.1 Identification of the Gh-rTDH purified from G. hollisae………...…….24 3.2 Gh-rTDH caused in vitro liver cell damage……….26

3.2.1 Gh-rTDH-FITC bound the margin of liver cells and invaded their

nucleuses………..29 3.2.2 Gh-rTDH caused liver cells death by cell apoptosis………....31

3.3 Liver damages in vivo were induced by Gh-rTDH………..34 3.3.1 Acute hemolytic status in vivo was caused by Gh-rTDH…….………...….36

3.3.2 Gh-rTDH damaged the functions of albumin synthesis in liver and triggered immune system………38 3.3.3 Gh-rTDH might not cause in vitro cardiotoxicity and nephrotoxicity…….38

3.3.4 Liver biopsy demonstrate that the damages located in the periportal area of liver……….…..39 3.3.5 Decrease and recovery in metabolism of livers in living animals evaluated

by 18F-FDG PET/CT scan….………...42 3.4 Fiber form of Gh-rTDH did not cause in vivo and in vitro hepatotoxicity.……..50

3.5 G. hollisae and E. coli-TOPO-tdh but not E. coli-TOPO causes in vivo

hepatotoxicity………..………...53

3.5.1 Liver damages in vivo were induced by G. hollisae and E.

coli-TOPO-tdh………..53 3.5.2 Decrease and recovery in metabolism of livers in mice evaluated by

18

F-FDG PET/CT scan………...……..53

3.5.3 Liver biopsy demonstrates that the damages were located in the periportal area of the liver……….……..……..54

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4. CHAPTER 4 DISCUSSION……….……62 5. CHAPTER 5 CONCLUSION AND FUTURE PERSPECTIVES…………...…..67

5.1 Analyze the relationships between chronic liver diseases and TDH...67 6. CHAPTER 6 EFERENCES………...72

ix

List of Figures

Figure 1 Influence of water temperature on the concentration of V. vulnificus in Gulf

Coast oyster meats………..………3

Figure 2 TDH from V. parahaemolyticus damaged the intestine……….….5

Figure 3 Model of heat-induced conformational change of TDH……….6

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……….……..8

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………..…...9

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…..………….…….…...9

Figure 7 18F-FDG and Isoflurane were treated to each mouse before scan started….20 Figure 8 Purification and characterization of the Gh-rTDH protein………..24

Figure 9 Tandem mass spectrum of the doubly charged tryptic peptide at m/z 1024.543 from SDS-PAGE of Gh-rTDH………..………...26

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………..27

Figure 11 The MTT assay of mouse liver cells……….……..28

Figure 12 The MTT assay of human liver cells………..…...…..29

Figure 13 Subcellular localization of Gh-rTDH………..………....…30

Figure 14 The liver cells were administrated with 1μg/ml of Gh-rTDH for 24 hours and performed with TUNEL assay were observed by confocal microscopy………...32

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…...33

Figure 16 Liver function evaluation after a single administration of Gh-rTDH..…...35

Figure 17 Gh-rTDH induces an acute hemolytic status……….…….…37

Figure 18 Gh-rTDH might not cause cardiotoxicity and nephrotoxicity…….……...39

Figure 19 Liver biopsy demonstrate that the damages located in the periportal area of liver……….….….41

Figure 20 Low levels of 18F-FDG uptake could be noted in mice fed with different dosages of Gh-rTDH and shock state was noted………..46

Figure 21 18F-FDG PET/CT scan for mice treated with Gh-rTDH……...…….…….47

Figure 22 In the mice fed with Gh-rTDH for 8 hours, they were administrated for 0.07 mCi 18F-FDG by tail vein injection and images taking were performed……….50

x

administration of fiber form of Gh-rTDH………51

Figure 24 The pathological images of liver parenchyma which obtained from liver

biopsy in mice were observed by microscopy at 200X magnification……….52

Figure 25 E. coli-TOPO did not cause abnormal liver functions………55 Figure 26 Acute liver injury could be noted in mice fed with G. hollisae……….….56 Figure 27 Acute liver injury could be noted in mice fed with E. coli-TOPO-tdh…...57 Figure 28 18F-FDG uptakes in livers were obviously decreased in the mice fed with G. hollisae……….………58

Figure 29 18F-FDG uptakes in livers were obviously decreased in the mice fed with E. coli-TOPO-tdh……….…….………58

Figure 30 E. coli-TOPO did not cause obvious liver injury. .……….………59 Figure 31 The ratios of liver/muscle 18F-FDG uptake levels in mice fed with

bacteria……….60

Figure 32 Liver biopsy (tissue harvested at the time of animal sacrifice) following

bacterial infection……….…61

Figure 33 Oysters are the major seafood productions of Lu-Kung and Wang Kong in

central Taiwan………..69

Figure 34 Each patient is suggested to receive the examination of liver ultrasound in

our cooperative EDs after exposure to TDH for 1, 42, 178 and 365 days…………...71

List of Table

Table 1 Vibrio species implicated as causes of human disease and number of deaths

1

Chapter 1 Introduction

1.1 Vibrio species caused diseases throughout the world

Diseases caused by different Vibrio species have been observed in large

populations throughout the world, particularly in Asia, the United States, and

Africa.(1-3) V. cholera and V. parahaemolyticus are the major etiological agents of

Vibriosis, which is associated with the ingestion of raw, undercooked, or contaminated

seafood (1, 2) Grimontia hollisae (previously named V. hollisae) has been frequently

reported to cause diseases in humans, including severe gastroenteritis, hypovolemia,

and septicemia following the consumption of shellfish or oysters.(4-6) Vibrio species

that are associated with human illness are listed in Table 1, together with Centers for

Disease Control and Prevention (CDC) data on the number of reported cases and

deaths in the United States. Previous studies reported that during warm summer

months, virtually 100% of oysters will carry V. vulnificus and/or Vibrio

parahaemolyticus (Figure 1); densities in US Gulf Coast oysters often exceed 104

organisms/g of oyster meat. Although Vibrios do not appear to affect oysters, some

species may be pathogenic to marine life.(1, 7, 8) Although V. parahaemolyticus has

always been recognized as an important enteropathogen and cause human diseases.

2

1990s. This increase has been noted in multiple countries, including Japan and the

United States, and appears to be associated with the appearance of a new clonal group

with pandemic potential.(1, 9)

Table 1 Vibrio species implicated as causes of human disease and number of deaths

associated with infection with these species.(1)

NOTE:++, Most common clinical presentation; +, neither rare nor most common

clinical presentation; (+), rare clinical presentation. aData reflect Vibrio infections

reported to the Centers for Disease Control and Prevention during 1999. Data are

from the 24 states that reported cases; for many of these states, reporting of Vibrio

3

Data were kindly provided by R. Tauxe, US Centers for Disease Control and

Prevention, Atlanta. bData include 4 cases associated with foreign travel. cThe 31

reported deaths are from a group of 75 cases for which data on death were available.

Figure 1 Influence of water temperature on the concentration of V. vulnificus in Gulf

Coast oyster meats. Each point represents the geometric mean of all observations

recorded within a 3°C temperature range.(8)

1.2 Grimontia hollisae cause diseases in human

G. hollisae (had ever been named V. hollisae) was recently and more frequently

4

septicemia after the consumption of shellfish and oysters.(1, 4-6, 10) G. hollisae was

ever a species of Vibrio and was firstly reported by Hickman et al.(11) Moreover,

Thompson et al. reported that V. hollisae strains (GenBank/EMBL accession nos

AJ514909-AJ514911) shared 99.5 % 16S rDNA sequence similarity, but had only

94.6 % similarity to their closest phylogenetic neighbor, EnteroVibrio norvegicus. 16S

rDNA sequence similarity of V. hollisae and Vibrio cholerae was only 91 %.

Therefore, these results suggest that V. hollisae should be placed into a novel genus as

G. hollisae.(12)This organism is reported to not usually grow on TCBS agar or

Mac-Conkey agar but does grow well on sheep blood agar and marine agar.(13)

However, the mechanism of morbidity caused by G. hollisae had not been well

addressed. Although some specific laboratory examinations have been shown to be

the way to detect G. hollisae but the incidence was still highly suspected to be

underestimated because of the detecting techniques were not very popular throughout

the world.(11, 13, 14)

1.3 Thermostable direct hemolysin was a violence factor

Thermostable direct hemolysin (TDH) was well known as a toxin constituted of

165 amino acid residues and which performed a variety of biological activities

5

Figure 2 TDH from V. parahaemolyticus damaged the intestine. (1) PBS was treated

to the intestine of rabbit and did not cause damages. (2) TDH from V.

parahaemolyticus damaged the intestine where lumens suffered edema and

hemorrhage.(15)

Some previous study reported that TDH is detoxified by aggregation into fibrils

after being heated at 60-70 °C, which can be reversibly refolded into the toxic native

form by being rapidly cooled after unfolding at higher temperatures (Figure 3).(16,

6

Figure 3 Model of heat-induced conformational change of TDH. Rapid heating and

cooling (A) slow heating and cooling (B).(16) TDHn: native TDH; TDHu: unfolded

TDH; TDHi: intermediate of TDH with fibrillar structure

The hemolytic activity of TDH is suppressed by the addition of Congo red, a

dye known to be sensitive to amyloid fibrils.(16, 17) These findings support the idea

that the conformational change in TDH, with the increase in β-sheet content, in a cellular membrane, may be associated with its cytotoxicity. The toxin effects of TDH

had been identified from a variety of Vibrio species, including V. cholera non-O1, V.

parahaemolyticus, V. mimicus, V. alginolyticus, and G. hollisae.(18-22) Among them,

the production of TDHs were reported to present with similar thd gene sequences and

7

but not in other Vibrio species.(18) In addition, the lipophilic effect of Gh-TDH is still

not clearly discussed in current reports. The toxic effects of TDH have been reported

to be mainly localized to the intestinal portion of gastrointestinal (15, 17, 24).

However, a relationship between TDH and the liver has not been reported or analyzed.

TDH might not only be absorbed by the intestine but may also cause secondary injury

to the liver via effects on the venous return of the portal system. In this study, we

aimed to analyze the hepatotoxicity of TDH using in vivo and in vitro analyses and to

provide evidence regarding the acute injury and recovery stages of THD-induced

8

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

hollisae

G.

TDH

Intestine

Portal

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 examination Cytoviability assay Confocal microscopy

The reason of cell death

Analyze the liver functions via withdraw

blood Analyze the cardiotoxicity and nephrotoxicity Liver biopsy 18_{F-FDG PET/CT} scan

10

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

11

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

12

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

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

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

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

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

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

18

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

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

18

F-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

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)

18

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

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

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

24

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

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

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

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

28

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

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

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

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)

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

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.

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

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

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

Gh-rTDH, their functions of albumin synthesis were damaged and were difficult to

recover in the initial 256 hours after exposure to Gh-rTDH. In addition, the globulin

levels were higher in groups fed with Gh-rTDH than in control group. This finding

indicated that Gh-rTDH might trigger immune system in circulation (Figure 16 E).

3.33 Gh-rTDH might not cause in vitro cardiotoxicity and nephrotoxicity.

The creatinine and CK-MB levels which significantly and clinically reflected

the kidney and heart injury were both not elevated in the groups fed with Gh-rTDH.

The levels of creatinine and CK-MB did not change in proportion to the feeding

dosages of Gh-rTDH. Moreover, the Troponin I levels were also normal in all mice

39

Figure 18 Gh-rTDH might not cause cardiotoxicity and nephrotoxicity.

Cardiotoxicity and nephrotoxicity were surveyed by withdrawing blood. Mice were

respectively fed with three different dosages of Gh-rTDH (1 μg, 10 μg and 100 μg) and control group was fed with PBS (each group n=5). Their levels of (A) creatinine

and (B) CK-MB were both normal in the serum of mice after single exposure to

Gh-rTDH.

3.34 Liver biopsy demonstrate that the damages located in the periportal area of

40

In the control group, the liver biopsy was performed and normal liver

parenchyma without pathological change was noted (Figure 19 A and B). However, in

mice fed with 10 μg of Gh-rTDH, the liver biopsy demonstrated the preserved liver parenchyma architecture with mild congestion over the periportal areas at the low

power magnification, and spotty liver cells damage around the portal vein at the high

power magnification. These damages were obviously located in the periportal area

(zone 1 of the liver acinus) (Figure 19 C and D). Moreover, in mice fed with 100 μg of Gh-rTDH, severe congestion with hemorrhage were noted at the low power

magnification and high power magnification revealed that confluent injury of liver

cells with intracytoplasmic acidophilic and ballooning change and nuclear pyknosis.

The periportal area damages of liver were more severe in mice fed with 100 μg than in mice fed with 10 μg of Gh-rTDH (Figure 19 E and F). Otherwise, the similar findings could be noted in each mice group received liver biopsy (each group n=3).

Therefore, these findings demonstrated that Gh-rTDH drained from the portal vein

41

42

liver. The pathological images of liver parenchyma which obtained from liver biopsy

in mice, were observed by microscopy at low power magnification -100X (A)(C)(E)

and at high power magnification- 400X (B)(D)(F). In the mice fed with PBS (control

groups), (A) the parenchyma were homogenous and health, (B) liver cells around

portal vein were not damaged. In the mice fed with 10 μg of Gh-rTDH, (C) the parenchyma were mild congestive over the periportal areas and (D) the spotty

damages could be noted in liver cells around the portal vein. In the mice fed with 100

μg of Gh-rTDH, (E) the parenchyma were severe congestive with hemorrhage around the periportal areas and (F) most of the liver cells around the portal vein were

damaged, these cells revealed that confluent injury of liver cells with intracytoplasmic

acidophilic and ballooning change and nuclear pyknosis. Above all, these images

demonstrated that the Gh-rTDH was absorbed by intestine and caused secondary

injury to the liver via venous return of portal system.

P: portal vein, Arrows: the damaged liver cells

3.35 Decrease and recovery in metabolism of livers in living animals evaluated by

PET/CT scan

In the mice fed with Gh-rTDH, the levels of 18F-FDG uptake were decreased

43

color light in merge images indicated that 18F-FDG was uptake by cells. General

lower level of 18F-FDG uptake could be noted in mice fed with higher dosage with

Gh-rTDH and shock state complicated with multiple organ failure was highly

suspected (Figure 20A-I). The level of 18F-FDG uptake in liver was further surveyed

in cross section images. In our study, each mouse had three series of images including

CT, PET and merge of CT and PET after receiving PET/CT scan (Figure 21A). We

found that in the mouse fed with Gh-rTDH, their 18F-FDG uptake in liver were

obviously fewer than in mouse fed with PBS, and their decreases were in proportion

to the dosages of Gh-rTDH (Figure 21B). Moreover, we also noted that the ratios of

liver/muscle 18F-FDG uptake level were obviously decreased in the 8th hour after

feeding with Gh-rTDH and the severity was dose-dependent. In addition, the ratios of

liver/muscle 18F-FDG uptake level recovered to the normal range and even crossed

the normal range in the 72th and 168th hour after feeding with Gh-rTDH, respectively.

These results indicated that the metabolisms of livers exposed to Gh-rTDH were

initially decreased and the recovery continued at least for one week after single

exposure of toxin (Figure 21C). The severities of organ damages were provided by

18

F-FDG PET/CT scan in the mice fed with Gh-rTDH for 8 hours. Intestine and liver

were both the major organs damaged by feeding with different dosages of Gh-rTDH

47

Figure 20 Low levels of 18F-FDG uptake could be noted in mice fed with different dosages of Gh-rTDH and shock state was noted. All mice had 3 series of images

including (A)(D)(G) CT, (B)(E)(H) PET and (C)(F)(I) merge of CT and PET. In the

8th (A-C), 72th (D-F) and 168th (G-I) hours after feeding with Gh-rTDH, the general

uptake of 18F-FDG were all decreased in proportion to the dosages of Gh-rTDH. Mice

in control group were fed with PBS.

Figure 21 18F-FDG PET/CT scan for mice treated with Gh-rTDH. Mice were administrated for 0.07 mCi 18F-FDG by tail vein injection and images taking were

48

performed 1 hour later. (A) All mice had 3 series of images including CT, PET and

merge of CT and PET. The location of liver was labeled as a dotted line where the

cross section images were obtained. (B) These images were cross section of livers. In

the 8th, 72th and 168th hours after feeding with Gh-rTDH, the uptake of 18F-FDG in

livers were all decreased in proportion to the dosages of Gh-rTDH. (C) The 18F-FDG

uptake value was calculated using ROI in each mouse (total n=60); the ROIs of liver

and muscle were recorded for semi-quantification in the groups fed with PBS

(control), 1 μg, 10 μg and 100 μg of Gh-rTDH (each group n=15, and each group were equally divided into 3 groups in different time points: 8th (n=5), 72th (n=5) and

168th (n=5) hours). The ratios of liver/muscle 18F-FDG uptake level were much fewer

in mice fed with Gh-rTDH than in those fed with PBS within the initial 8 hour.

Moreover, the levels returned and crossed to the normal range in the 72th hour and

50

Figure 22 In the mice fed with Gh-rTDH for 8 hours, they were administrated for

0.07 mCi 18F-FDG by tail vein injection and images taking were performed. The

severities of organ damages were provided by 18F-FDG PET/CT scan. Intestine and

liver were both the major organs damaged by feeding with (A) 1 μg; (B) 10 μg and (C) 100 μg of Gh-rTDH and the damages were also dose-dependent.

3.4 Fiber form of Gh-rTDH did not cause in vivo and in vitro hepatotoxicity

The fiber form of Gh-rTDH did not have hemolytic activity in our study. Cell

morphology did not change after exposure to fiber form of Gh-rTDH for 24 hours and

MTT assay revealed that the cytoviability of FL83B cells also did not decrease after

treating fiber form of Gh-rTDH in different dosage and treating durations. In mice fed

with fiber form of Gh-rTDH their liver function tests were normal (Figure 23A-E) and

51

Figure 23 The levels of liver functions were normal in the serum of mice after single

administration of fiber form of Gh-rTDH. Six-week-old female BALB\c were

52

10 μg and 100 μg) and control group were fed with PBS (each group n=5). The levels of (A) GPT and (B) GOT did not elevate. (C) Hyperbilirubinemia and (D)

hypoalbumenia were not occurred. (E) The levels of globulin were not increased after

exposure to Gh-rTDH.

Figure 24 The pathological images of liver parenchyma which obtained from liver

biopsy in mice were observed by microscopy at 200X magnification. In the mice fed

53

liver cells around portal vein were not damaged. Similar finding were noted in the

mice fed with (B) 10 and (C) 100 μg of fiber form of Gh-rTDH. Above all, fiver form of Gh-rTDH did not cause periportal area damages in liver. P: portal vein

3.5 G. hollisae and E. coli-TOPO-tdh but not E. coli-TOPO causes in vivo hepatotoxicity

3.51 Liver damages in vivo were induced by G. hollisae and E. coli-TOPO-tdh.

In the control group, the GOT and GPT level did not elevate after feeding them

with a single dosage of E. coli-TOPO (Figure 25). However, in the group fed with G.

hollisae (Figure 26) and E. coli-TOPO-tdh (Figure 27), the mean GOT and GPT

levels were obviously elevated and the highest levels could both be respectively found

in the 16th hour and 8th hour after feeding with bacteria. Higher concentrations of

bacteria are clearly associated with higher levels of GOT and GPT, which clinically

indicate more severe liver injury in mice. Acute hemolytic status, poor functions of

albumin synthesis and more triggered immune system could be noted in the mice fed

with G. hollisae and E. coli-TOPO-tdh. These patterns were similar with mice directly

fed with toxin (Gh-rTDH).

54

PET/CT scan.

We found that in the mice fed with G. hollisae (Figure 28) and E.

coli-TOPO-tdh (Figure 29), 18F-FDG uptakes in their livers were obviously fewer

than in mice fed with E. coli-TOPO (Figure 30), and their decreases were in

proportion to the amount of bacteria. Moreover, we also noted that the ratios of

liver/muscle 18F-FDG uptake levels decreased in the 8th hour after feeding with G.

hollisae and E. coli-TOPO-tdh. Among them, the ratios of liver/muscle 18F-FDG

uptake levels recovered in the 72th and 168th hour, respectively (Figure 31A and B).

Moreover, E. coli-TOPO did not cause significant liver injuries (Figure 31C).

3.53 Liver biopsy demonstrates that the damages were located in the periportal area

of the liver.

In mice fed with G. hollisae, the liver biopsy demonstrated obvious cell

damages over the periportal areas (Figure 32A). Spotty liver cell damages around the

portal vein were noted in mice fed with E. coli-TOPO-tdh (Figure 32B). Finally, in

the mice fed with E. coli-TOPO, there was no pathological damage in liver

55

Figure 25 E. coli-TOPO did not cause abnormal liver functions. In the control group,

the GOT and GPT level did not elevate after feeding them with a single dosage of E.

56

Figure 26 Acute liver injury could be noted in mice fed with G. hollisae. GOT/GPT

levels were obviously elevated and the highest levels could both be respectively found

57

Figure 27 Acute liver injury could be noted in mice fed with E. coli-TOPO-tdh.

GOT/GPT levels were also obviously elevated and the highest levels could both be

respectively found in the 16th hour and 8th hour after feeding with bacteria. This

58

Figure 28 18F-FDG uptakes in livers were obviously decreased in the mice fed with G. hollisae. The decreases were in proportion to the amount of bacteria.

Figure 29 18F-FDG uptakes in livers were obviously decreased in the mice fed with E. coli-TOPO-tdh. The decreases were in proportion to the amount of bacteria.

59

Figure 30 E. coli-TOPO did not cause obvious liver injury. The uptakes of 18F-FDG were not decreased.

60

Figure 31 The ratios of liver/muscle 18F-FDG uptake levels in mice fed with bacteria. The ratios of liver/muscle 18F-FDG uptake levels recovered in the 72th and 168th hour,

61

respectively (A and B). Moreover, E. coli-TOPO did not cause significant liver

injuries (C).

Figure 32 Liver biopsy (tissue harvested at the time of animal sacrifice) following

bacterial infection. In mice fed with G. hollisae, the liver biopsy demonstrated

obvious cell damages over the periportal areas (A). Spotty liver cell damages around

the portal vein were noted in mice fed with E. coli-TOPO-tdh (B). Finally, in the mice

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Chapter 4 Discussion

In this study, liver cells were firstly treated with G. hollisae TDH and the in

vitro hepatotoxicity was demonstrated by direst observation and MTT assay. The

hepatotoxicity caused by Gh-TDH was both dose- and time-dependent. Very low

concentration of TDH (> 10-6 μg/ml) damaged the liver cells. We also noted that the

MTT assays yielded a similar pattern over 12, 16, 24, and 48 hr under different toxin

concentrations. One possible explanation is that when the concentration of toxin

increased, cells were not only killed by this toxin but also probably suffered cell

division suppression. Therefore, when we prolonged the treatment durations, the

number of surviving cells did not clearly differ between the 4 time points. Naim et al.

reported that V. parahaemolyticus TDH caused Rat-1 cells injury and the TDH might

induce cytotoxicity by acting inside the cells.(27) In our study, we noted that the

Gh-rTDH-FITC invaded inside of liver cells via binding around the margin of cell and

further located in their nucleus in short time. Therefore, the destructions caused by G.

hollisae TDH were quite quick and lethal in liver cells.

Morros et al. firstly reported a patient suffered G. hollisae infection and

presented with liver cirrhosis and hepatic encephalopathy in 1982.(28) Some previous

studies demonstrated that the symptoms of patients with Vibrio species infections

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However, the evidence of hepatotoxicity caused by G. hollisae TDH had never been

provided. In this study, we found that the GOT and GPT levels of mice fed with

Gh-rTDH were obviously increased and acute liver injury were highly suspected.

Muscle injury was not favored as the major reason for the elevation of GOT/GPT, as a

18

F-FDG PET/CT scan did not show muscle injury. Moreover, the liver biopsy

revealed that the periportal areas of liver were damaged and their severity associated

with the dosage of Gh-rTDH. The functions of periportal area in liver were well

known as oxidative energy metabolism of fatty and amino acids, glucose release and

glycogen formation, ammonia detoxification, protective metabolism, and the

synthesis of albumin.(29-31) Therefore, mice with periportal area injury caused by

Gh-rTDH might also suffer some complications including malnutrition, protective

system destruction, hepatic encephalopathy and hypoalbumenia. Clinically, the

hypoalbumenia could be induced by decreased (hepatic) production or increased loss

(gut tract loss). The result of hypoalbumenia in this study might be influenced by both

mechanisms. The globulin levels also increased as the protective systems of the livers

were damaged and the toxin triggered their immune systems in the circulation.

Overall, the evidence of hypoalbumenia could be provided in our mice fed with

Gh-rTDH and in whom their globulin levels also increased because of their protective

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TDH was well known as having a strong hemolytic activity in vitro.(14, 23, 32,

33) However, the in vivo hemolysis caused by TDH had not been well provided. The

acute hemolytic status in vivo results in acute anemia, which would exacerbate tissue

hypoxia and organ hypoperfusion. Therefore, septicemia caused by Vibrio species

with the tdh gene might be more critical than that caused by the Vibrio species

without the tdh gene. Clinically, the hepatotoxicity might be caused via hemolysis.

However, the pathological findings revealed that the hepatic injury was mainly

located at the periportal areas, and the injury was not diffused. It is suspected that the

major etiology is toxin absorption and injury to the liver via the venous return of the

portal system.

Clinical PET/CT scan had been reported as an excellent tool to survey tumor and

organ metabolism in small animals.(34) Damages of liver caused by Gh-rTDH could

be demonstrated by blood withdrawal and liver biopsy. However, the conditions of

recovery and organ metabolism in living animals were difficult to be analyzed.

Therefore, 18F-FDG PET/CT scan was performed in our assessment. We noted that the

uptake of 18F-FDG in the livers decreased in proportion to the feeding dosages of

Gh-rTDH, which indicated that the damages of livers in living animals were

dose-dependent. After single exposure to Gh-rTDH, the uptake of 18F-FDG gradually