MBO8T1

June 8, 2007

Research Report

Biological Effects of TOFIX-S

Using Intratracheal Instillation

Professor Isamu ak

Institute of Industrial Ecological Sciences

University of Occupational and Environmental

Health, Japan

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Table of Contents

Chapter 1 Research Project and Research Team

Chapter 2 Research Objectives

Chapter 3 Research Design and Methods

Chapter 4 TOFIX-S Particle Characteristics

4.1 Contents of fibrous materials

4.2 Fiber diameters and lengths

Chapter 5 The Recovery of TOFIX-S from the Animal Tissue

5.1 Recovery procedures

5.2 Recovery rates

Chapter 6 Intratracheal Instillation

6.1 Instillation study

6.2 Protocol

Chapter 7 Results of the Intratracheal Instillation

7.1 Rat growth curves

7.2 Wet weights of the organs at each time point of sacrifices

7.3 Quantification of lung burdens of TQFIX-S

7.4 Biopersistence of TOFIX-S from the lungs (biological half

time)

7.5 Formation of 8-hydroxydeoxyguanosine in lung tissue

7.6 Expression of gene related to inflammation and fibrosis in

lung tissue

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7.7 Histopathological examination

Chapter 8 Discussion

8.1 Rat growth curves

8.2 .We-t organ weights of the rats at each time point .of.

sacrifices

8.3 Biopersistence of TOFIX-S

8.4 8-hydroxydeoxyguano sine

8.5 TIMP-2

8.6 Histopathological changes

Chapter 9 Conclusion

8 Figures

3 Tables

7 Micrographis

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Chapter 1 Research Project and Research Team

Research Project

Biological Effects of TOFIX-S Using Intratracheal Instillation

Research Team

Lead investigator:

Professor Isamu Tanaka, Dr. Eng.,

Department of Environmental Health Engineering,

Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Japan

(Graduated from the Department of Engineering, Kyushu University in 1967)

Researchers involved in the project: -

Professor Yasuo Morimoto, MD, DMS,

Department of Occupational Pneumology,

Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Japan

(Graduated from Kagoshima University School of Medicine in 1986)

Associate Professor Toshihiko Myojo, Dr. Eng.,

Department of Environmental Health Engineering,

Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Japan

(Graduated from Department of Engineering, Kanazawa University in 1977)

Associate Professor Akira Ogami, MI), DMS, ______________

Department of Occupational Pneumology,

Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Japan

(Graduated from University of Occupational and Environmental Health Graduate School

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of Medicine in 1997)

Assistant Professor Takako Oyabu, DM3, 7 rt

Department of Environmental Health Engineering,

Institute of Industrial Ecological Sciences,

University of Occupational and Environmental Health, Japan

(Graduated from the Department of Science, University of Kyushu in 1983)

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Chapter 2 Research Objectives

The aim of this study is to assess the potential biological hazards of TOFDC-S when they enter the

body, especially into the lung, by determining the biopersistence of TOFIX-S in the lung and

biological reactions including histopathological examination and expression of genes which are

related to fibrogenesis and carcinogenesis.

It is known that certain types of dust cause respiratory diseases when enter the alveolar region

where gas exchange takes place but the effects and degree of the -diseases vary greatly according to

the dust type. For pulmonary fibrosis (pneumoconiosis) and canoers in the lungs, -it is postulated

that the longer retention of dust in the alveolar region increases the length of time of dust

contacting the body, resulting in the induction of various kinds of biological reactions that may

progress to fibrosis and cancers.

In this study, examination of TOFJX-S with electron microscopy is first made to determine the

shapes of TOFIX-S. Then, we conducted an experiment in which TOXIF-S are intratracheally

instilled in rat lungs and the amounts of TOFlX-S retained in rat lungs are quantified by means of

serial sacrifices to determine the rate of lung clearance of TOFIX-S. Histopathological examination

and cytokines which are responsible for fibrotic and carcinogenic changes in the lung are examined

to assess the biological effects of TOFDC-S.

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Chapter 3 Research Design and Methods

The biopersistence of chemicals that are introduced into the lung via airways is vital to assess their

health effects or biological effects in humans.

The fate of chemical substances in the lung depends largely on their physiochemical characteristics,

dimension and especially biopersistance in the lung, over time, that is involved directly with the

length of time required for the initiation of biological responses and the effects of such responses.

The significance of this understanding has begun to be appreciated in recent years and, therefore,

biopersistence of fibrous dust which contributes especially to fibrogenesis and carcinogenesis in the

lung have being investigated as the most important parameter.

As gene expression of cytokines which is an obvious potential for mediating fibrosis and

carcinogenic changes is related to the response mechanisms by which the chemical produces final

conditions, the measurement of gene expression of cytokine is a widely used tool to deteimine

fibrotic and carcinogenic changes that occur in the lungs. On this basis, we planned and conducted

an experiment according to the following procedures to obtain our objective:

I. Identify the geometric shapes of TOFIX-S by electron microscopy

2. Conduct an animal experiment in which TOFDC-S is instilled intratracheally into rats

3. Decide which method to use for quantification of TOFIX-S entered in the animal lung

4. Quantify TOFIX-S retained in the lungs to determine the rate of lung clearance by means of

serial sacrifices of a given number of rats at certain time points

5. Measure the amount of cytokine which is deeply involved in fibrotic or carcinogenic changes

in the lungs;

6. H1istopathological examinations of the lungs

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Chapter 4 TOFIX-S Particle Characteristics

4.1 Contents of fibrous materials

Filters containing TOFIX-S and deposited platinum on them were prepared for scanning -electron

microscopy assessment of TOFTX-S lengths and diameters; and, subsequently digital micrographs

were made (Micrograph 1).

Micrograph 1 A scanning electron micrograph of TOFIX-S

There are particulate matter and fibrous shaped particles fibrous shaped particles having a

length-to-diameter ratio greater than 3:1. Particles with a diameter of more than 0.2jpm in the

randomly selected 20 fields of view of the scanning electron microscope were counted. The number

of fibrous particles and non-fibrous particles quantified by the scanning electron microscopy were

124 and 1,136, respectively. This indicates that the number of fibrous particles accounts for 9.8 %

of total number of particles counted. Since a product that contains asbestos exceeding 0.1 % by

weight which asbestos bears to the whole product is defined as an asbestos product according to the

Ordinance on the Prevention of Hazards due to Asbestos, TOFJX-S can be defined as an fibrous

product, therefore, we though that it is relevant to assess the potential hazards of TOFDC-S.

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4.2 Fiber diameters and lengths

Diameter and length are the basic determinants of fibrous materials. TOFIX-S was collected On the

filter and, then, platinum was deposited on the filter and an image was taken using a scanning

electron microscope. Computer-assisted measurements using NIH image were made from scanned

SEM micrograph for diameters and lengths of the 321 TOFIX-S. The results were plotted on a

logarithmic-normal probability paper as shown in the Figures 1 and 2.

98-

5a

'0-

-9 a

UU

945g

a0-

- 7 U

10 U

US90U

5U

7010

207 ~Pg 

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Both the distribution of diameter and length were found to be a logarithmic-normal probability

distribution fitted in an approximate linear curve on the logarithmic-normal probability paper For

fiber diameter, the geometric mean diameter and geometric standard deviation of the fibers were

1.4 im and 1.5, respectively. For fiber length, the geometric mean length and geometric standard

deviation of the fibers were 6.2 pmn and 1.8, respectively Approximately fifteen percent of the

fibers were found to be less than 0.8 in. in diameter but were shorter than 20 pim. It is well known

that for asbestos the tinner and longer the fibers (over 20 pm in length), the greater the hazardous

potential; no fibers longer than 20 pm were found for TOFIX-S.

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Chapter 5 The Recovery of TQFLX-S from the Animal Tissue

5.1 Recovery procedures

In order to measure TOFIX-S satisfactorily: to obtain excellent recovery rates, we examined

procedures, including pretreatment conditions and instrument suitable for quantitative analysis of

TOF]X-S present in the animal tissue, to determine lung burdens.

The samples to obtain recovery rates were prepared by first taking a precise amount of TOFDC-S'

and adding it to the lung tissue (liver was used in the recovery procedures because it provides us a

larger amount of samples), and then a method to recover the -given amount of TOFIX-S from each

sample by using several ashing techniques was studied. A quantification method used in this study

has the combined advantages of suitability of pretreatment and analytical conditions and

instruments and, therefore, will provide us with excellent recovery rates. The procedures are shown

in Figure 3.

5.2 Recovery rates

TOFDX-S of either 0, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 mg was added to each I g of homogenized liver

and each placed in a glass bottle. Three preparations each were made, making 21 samples in total.

For the quantitative analysis of TOFT.X-S in the lung tissue samples was made following the

procedures, as shown in Figure 3, to obtain recovery rates.

The amount of TQFDC-S in each sample which -contained 50.1 % Ti was calculated by converting

measured Ti concentrations. The results are shown in Table 1.

The results showed that almost 100% of TOFIX-S added to each sample was recovered, therefore,

we concluded that our quantification procedures could be used as a method to quantify the lung

burden of TQFIX-S in this intratracheal instillation study.

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Lungs

Freeze dhying

Low-temperature ashing]

Filtering

2.5m1 Acid sol.*

Microwave wet asbing (Milestone Ethos Type 1600)

Digesting conditionsC250W 3min

500W 20mm J

Preare tocontan voumeof50m

Quantify Ti amount (ICP-AES analysis)

* (1O0ml+40g) H2SO4+(NH4)SO 4

Figure 3 Procedures for the quantification of TOFLX-S lung burdens

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Table 1 The results of recovery rates

TOFIX-Sqanifd ~IOM~D-S added Rawyrates Mon

C-1 427200 < 0.001

C-2 416000 < 0.001

C-3 416000 < 0.001

C+0.O5mg-1 1469000 0.0505 0.05 101.1

C+0.05mg-2 1463000 0.0503 0.05 100.5

C+0.O5mg-3 1466000 0.0504 0.05 100.8 100.8

C-Ilmg-1 2410000 0.0956 0.10 95.6

C+0.lnig-2 2573000 0.1034 0.10 103.4

C+0. lmg-3 2539000 0.1018 0.10 101.8 100.3

C+0.2mg-1 4792000 0.2098 0.20 104.9

C+0.2ing-2 4645000 0.2027 0.20 101.4

C+0.2mg-3 4716000 0.2061 0.20 103.1 103.1

C+0.5m-1 11130000 0.5134 0.50 102.7

C+0.5mg-2 9850000 0.452 1 0.50 90.4*

C+0.5mg-3 11140000 0.5139 0.50 102.8 102.8

C+1.Omg-1 21650000 1.0175 1.00 101.8

C+1.Omg-2 21530000 1.0118 1.00 101.2

C+1.Omg-3 21920000 1.0304 1.00 103.0 102.0

*Excluded from the data due to an error in measurment

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Chapter 6 Intratracheal Instillation

6.1 Instillation study

Potential hazards of chemical substances in human differ significantly with the routes of exposure

(oral, skin and respiratory tracts), biological effects of such. chemicals in humans need to be

assessed in animal studies by using routes through which human exposure occur. Human contact

with TOFIX-S generally takes the following form: they are inhaled with air and may possibly enter

the body through respiratory tracts. For this reason, biological effects should be available from

animal studies in which inhalation would be the most practical study design for this purpose.

However, since it requires certain equipment and considerable cost and time, intratracheal

instillation whose route or exposure is similar to inhalation has been widely used as an alternative

exposure procedure. A method was developed to administer accurate intratracheal instillation of test

materials in the animal lung. A given amount of test particles suspended in a solution is injected by

syringe through the cannula into the trachea, delivering the test particles directly into the lungs.

Regarding the advantages and disadvantages of the method, intratracheal instillation administers an

accurate dosage of materials but it has the disadvantages that large amount of dose in quantity is

delivered at one time and a solution in which test materials are suspended is also administered in

the lungs. However, the route of introduction into body is an unnatural route of exposure in human

exposure, but the method does provide useful information on how the test particles behave in the

lungs.

The main theme of this study is to estimate the biological effects of TOFIX-S. For this purpose, we

conducted an intratracheal instillation experiment to examine the biopersistence of TOFLX-S in the

lungs in relation to its lung clearance.

6.2 Protocol

6.2.1 Experimental animals

Nine-week-old male Wistar rats were used.

6.2.2 Number of rats

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Seventy rats were randomly divided into two similarly sized groups:

Intratracheal instillation group: TQFIX-S exposing group

Control group: Physiological saline exposing group

6.2.3 Administratiion of TOFIX-S

TOFIX-S was suspended in physiological saline at the rate of 2 mig per 0.4 ml and administered to

35 rats by a single intratracheal instillation into the under ether anesthesia. Rats in the control group

were given only the 0.4 ml of physiological saline.

6.2.4 Time points for sacrifices after instillation

Rats were sacrificed at the following time points after intratracheal instillation:

- 3 days after instillation 10 rats per group were sacrificed.

- 1 week after instillation, 5 rats per group were sacrificed.

- 1 month after instillation 5 rats per group were sacrificed.

-3 months after instillation 5 rats per group were sacrificed.

-6 months after instillation 10 rats per group were sacrificed.

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Chapter 7 Results of the Intratracheal Instillation

7.1 Rat growth curves

The growth curves of the rats were recorded every week throughout the experiment The results are

shown in Figure 4.

700

600

S 500

S 400

- 3001- Saline instilled group/Control group

100

0

5 10 i5 20 25 30 35

Time (age in weeks)

Figure 4 Growth curves.

No difference was found in rat growth curves (weight changes) between two groups.

Behaviors and health conditions of rats in the cages were also observed every day; there was

nothing particular to be noted.

7.2 Wet weights of the organs at each time point of sacrifices

The results of the measurement of the wet weights of rat lung, liver, kidney and spleen at each time

point of saciffices are shown in Table 2.

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Table 2 Wet weights (g) of rat lung, liver, kidney and spleen at each time point of sacrifices.

Lung Liver Kidney Spleen

After 3 days

Control group 1.26±0L.07 15.3±1.3 2.34±0.15 0.85±0.12

TOFsflted grou 1.29±0.22 14.4±1.3 2.30±0.18 0.82±0.12

After 1 week

Controligroup 1.30±0.09 15.8±1.3 2.48±0.27 0.78±0.14

TOFDX-5 insWdflatc p 1.28±0O.06 15.7 ±0.7 2.50:E0. 14 0.86±0.08

After 1 month

Control group 1.36±0.05 18.0±11.7 2.87±0.19 0.97±0.08

T~XS bmWat group 1.36±0.04 17.3±0.8 2.74±0.06 0.91±0.08

After 3 months

Controlgroup 1.39±0.12 15.8±1.7 2.75±0O.28 0.85±0.06

TOMD-S hwd&W tgrcup 1.50±0.09 17.2±1.6 2.81±0.13 0.91+--0.16

After 6 months

Controlgroup 1.61±0O.06 19.4±2.5 3.32±i0.27 0.954±0.13

TOFD-S insfillaW goup 1.53±0.10 19.74±3.0 3.24±0.37 0.96±0L.12

No significant difference in wet weights of lung, liver, kidney and spleen at each time point of

sacrifices were found in the two groups.

7.3 Quantification of lung burdens of TOFIX-S

1) Method for quantification of TOFIX-S in the lungs

Five rats in each group were sacrificed at given time points to quantify the amount of TOFIX-S

retained in the lungs following the procedures described in Figure 3. The amount of TOF1X-S in

each sample which contained 50.1 % Ti was calculated by converting measured Ti amounts.

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2) Lung burden of TOFIX-S

The results of measurements are shown in Table 3.

Table 3 Lung burden of TOFJX-S at each time point of sacrifices

Lung burden of TOFDC-S (mg)

3 days after instillation 1.21 ±0.28

1 week after instillation 0.73 ±0.22

1 month after instillation 0.71 ±0.15

3 months after instillation 0.32 ±0.11

6 months after instillation 0. 17 ±- 0.06

Ti amounts in rat lungs of the control groups were found to be extremely low (less than 1 pg) when

compared with to those in rat lungs of the TOFIX-S instillated group, those concentrations were

regarded as zero in quantifying the amount of TOFIX-S in the lungs.

It was found that the lung burden of TOFIX-S decreased with time after instillation.

The dose of TOFD(-S was 2 mg and it decreased to an average of 1.21 mg at three days after

instillation, suggesting that particles deposited in the tracheobronchial airways were cleaed.

It is known that particles deposited in the tracheobronchial airways are borne away by the

continuously upward heaving mucous layer; most of them are then exhaled in about one day. Other

particles retained in the lung are thought to be penetrated more deeply get into the pulmonary

alveoli, which is a collection of approximately 3 X 1 08 alveoli of tiny air sacs with a surface are of

about 200 pm, having a total surface area of about 100 square nilimeters. Pulmonary alveoli are

surrounded on the outside with fine blood vessels. It is in the alveoli in the lungs that gas exchange

takes place as the vessels absorb oxygen from the alveolus through its thin-wall. Since the alveoli

in the lungs are lined by epithelial cells without cilia, in the case of inhaled foreign substances the

alveoli dissolve the substances by its fluid and clear them or produce macrophages to phagocyte

and digest such substances to clear. Such substances phagocytosed by these macrophages are

cleared as some transported to bronchial regions lined by ciliated epithelial cells or some get into

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the lymphatic system.

The values shown in Table 3 may indicate that TOFDC-S deposited in the alveolar region was

cleared from the lungs by these clearance mechanisms.

7.4 Biopersistence of TQFIX-S from the lungs (biological half time)

Relationship between the lung burden of TOFIX-S at each given time point after instillation were

plotted on semi-logarithmic graph to estimate the rate of TOFIX-S pulmonary clearance from the

measurements shown in Table 3. The results are indicated in the form of a filled rhombus mark

10.0

Biological half time: 12 months IPTZS

Biological half time: 2.2 months

0.1 -I I __ - I

0 1 2 3 4 5 6

Time points after instillation (month)

Figure 5 Changes of lung burden of TOFIX-S and PTlI with time

The results obtained in our laboratory for the changes of lung burden of potassium octatitanate

whisker (PT 1) with time are indicated in Figure 5 in the form of an open rhombus mark (C>) for

reference.

The lung clearance rate of TQFDC-S can be expressed as the biological half time of TOFIX-S

retained in the rat lungs that can be obtained by lung burdens of TOFIX-S measured at three days, 1

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week, 1 month, 3 months and 6 months after instillation (Co). This can be summarized by the

formula:

dC/dt =-kC(1

Initial conditio: t0, C =Co (2)

where the lung clearance rate of TOFIX-S is assumed to be proportional to the lung burden of

TOFIX-S (C) and k is a lung clearance rate constant (1/day) which can be obtained by using the

results of the experiment

Then, the following equation is obtained when Equation (1) is solved using the initial condition (2):

C/Co-- EXP(-kt) (3)

The term biological half time (Tit2) refers to (is defined as) the time by which half of the

administered amount of particles is cleared from the lung. The T1t2 is given by using the following

Equation (4):

Ti2= 0.693/k (4)

The biological half time of the lung burden of TOFDC-S calculated by using the measured lung

burdens was 2.2 months. The Tin can also be assumed from the gradient of the line shown in

Figure 3. T1/2 for PTl (potassium octatinate wisker) is 12 months.

7.5 Formation of 8-hydroxydeoxyguanosine in lung tissue

8-hydroxydeoxyguanosine which is formed when hydroxylation of deoxyguanosine at the C-8

position occurred is a major product of oxidative DNA damage and may induce lung injury and

carcinogenesis.

Up to the present time, 8-hydroxydeoxyguanosine was found in the cases in which lung injury was

produced due to environmental exposure to substances such as tobacco, asbestos and diesel exhaust

emissions; and a relationship between 8-hydroxydeoxyguanosine and lung cancer has been

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reported.

7.5.1 Measurement method

1) Flush a 1.5 ml centrifuge tube with nitrogen,

2) Put the lung tissue in the tube and add 1 ml Lysis solution,

3) Hold the tube on the ice for homogenization,

4) Centrifuge at 10,000 g for 20 seconds,

5) Discard the supernatant and add I ml Lysis solution aan

6) Centrifuge at 10,000 g for 20 seconds,

7) Discard the supernatant and add 180 id Enzyme Reaction solution,

8) Add 2 ml RNase and allow the reaction mixture to react in a constant-temperature bath at

50-C for 10 minutes,

9) Add 10 0d Proteinase solution and store in the constant-temperature bath at 50*C for 60

minutes,

10) Centrifuge at 10,000 g for 10 minutes,

11) Discard the supernatant,

12) Add 0.3m1 NaI solution,

13) Add 0.5m1 isopropyl alcohol,

14) Centrifuge at 10,000 g for 10 minutes,

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15) Discard the supernatant,

16) Add 1 ml A (wash fluid) and wash by centrifugation,

17) Add 1 ml B (wash fluid) and wash by centrifugation,

18) Drying

19) Add 0.1 imI1mMIEDTA, 1J4 I 2M of sodium acetate buffer solution, 4 0d of 5mg/mi nuclease

P1, and 2 W. of phosphoric acid; and incubate at 37 00 for 30 minutes,

20) Perform ion-exchange resin and centrifuge,

21) Transfer the solution to a tube with a filter, centrifuge and extract the solution,

22) Inject 20 W. DNA solution in the 1HPLC detector (electrochemical detector, ESA, Coulochem,

USA).

23) Use the 8OHdG/dG ratio to quantify 8OHdG.

With the 8OHdG/dG ratio, the amount of 8OHdG can be determined.

7.5.2 Measurements of 8-hydroxydeoxyguanosine

The levels of 8-hydroxydeoxyguanosine measured in the lungs of rats sacrificed at 3 days and 6

months after finstillation are shown in Figure 6.

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3.0 - E Control group

UTOFIX-S instiliated group 2.5

2.0

~1.5

1.0

0.5

0.0

3 days after instillaton 6 months after instilton

Figure 6 The levels of 8OHdG in the lungs of rat sacrifices at 3 days and 6 months after instillation

No significant differences were found between the instilled group and the control group for the

levels of 8OHdG both at 3 days and 6 months after instillation.

7.6 Expression of gene related to inflammation and fibrosis in lung tissue

7.6.1 Fibrosis-related gene

Type I collagen, Matrix metalioproteinases (MM~s) and Tissue inhibitors of metalloproteinses,

(TIMPs) are the genes involved in the production and metabolism of collagen. Collagen is deeply

involved in pulmonary fibrosis. Extensive deposition of collagen is thought to be the most

significant condition for pulmonary fibrosis and presumably depends on a balance between

synthesis and degradation of collagen.

7.6.1.1 Excessive production of collagen

Collagen in lung tissue is primarily of Type I collagen and it has been postulated that the extensive

deposition of Type I collagen induces fibrosis. The occurrence of excessive production of collagen

could be determined from examination of whether gene expression of Type I collagen increased.

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7.6.1.2 Regulation of collagen degradation ?l0T

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (MMPs) are

matrix-degrading enzymes. Deposition of collagen is regulated by a balance between MM~s and

TIPS. Abnormal degradations of collagen frequently occur when there is an imbalance between

MMW and TRP An imbalance of MM~s and MIN~ has been reported to result in

metalloprotemnase activation or inactivation, and relatively higher levels of TIPs than of MM~s

may contribute to the downregulation of degradation resulting in the deposition of collagen A late

stage or fibrotic stage in the process of fibrosis, the gene expression of TRAPi is elevated.

Conversely, when the level of MIMP activation is higher than that of TIMvIP, the degradation of

collagen is facilitated, which in turn results in lesser deposition of collagen. This is seen in an early

stage or lung injury and inflammation in the process of fibrosis and also seen in pulmonary lung

masses that can be attributed to cigarette smoking.

The mechanism of fibrosis caused whether by increased production of collagen or by regulated

degradation of collagen can be elucidation by means of quantification and analysis of the results.

It seems a reasonable procedure to first conduct a screening for expression of these genes, then, if

significant changes are found in any of these three genes, expression of the other two genes are

further examined. We decided to first measure the activation of TIMP and, if significant differences

in the levels of 'lIM were found compared to the control group, then further measure the levels of

'Type I collagen and MMP.R

7.6.2 Measurement method

7.6.2.1 RNA extraction procedures

Lung tissue was homogenized with 1.2 ml of Solution D. RNA was extracted from the lung tissue

by using a guanidium chiocyanate-phenol-chloroform extraction technique.

RNA extraction from lung tissue:

1) Add 1.2 ml of Solution D, 120 jpl of 2M Sodium Acetate (p114.0), 1.2 ml of Phenol DEPC

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1120 and 120 p1of Chioroform-Isoamyl alcohol (49:1) to the homogenized solution,

2) The mixture is agitated for 10 seconds and held on the ice.

3) Centrifugate at 15,000 rpm for 20 minutes at 400,

4) Inject the supernatant in the PHASE LOCK GEL(PLG) tube and centrifugate at 8,000 rpm for

5 minutes,

5) Transfer the resulting supernatant to a new centrifuge tube, mix with 1.2 ml of isopropanol

and hold at -20'C for 15 minutes,

6) Centrifugate at 15,000 rpm for 20 minutes at 4*C,

7) Discard the supernatant and add 360 p.1 of Solution D to the pellet,

8) Add 360 0. of isopropanol and hold at -80'C for 15 minutes,

9) Centrifuge at 15,000 rpm for 10 minutes at 40C,

10) Discard the supernatant, mix with 0.5 ml 75% ethanol and centrifuge at 15,000 rpm for 10

minutes at 40C,

11) Again mix with 0.5 ml of 75% ethanol and centrifuge at 15,000rpm for 10 minutes at 4*C,

12) Discard the supernatant and centrifuge for 5 minutes while sucking,

13) Sufficiently dry the RNA pellet and add TE 20-50 W. to dissolve the pellet,

14) Adjust the concentration to 2.2 pgg/pil by using spectrometer,

After extracting the RNA, gene expression of TRAvP was measured by perform reverse-transcription

polymerase chain reaction (RT PCR).

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7.6.2.2 RT (Reverse transcriptase)

1) Mix 5 x RT buffer, d-NTP, Randam hexamer(RB), RNAse inhibitor (RDI, Riverse

transc'riptase (RT) to 75.5 jlin total in a 1.5 ml eppendorf tube, add a sample of 10 ~ of

tissue (contains 22 gg of RNA), and dilute the mixture with 114.5 RI of DEPC H20 to 200 jI

in total.

RT-mixture:

5 XRT buffer 40 ~

d-NTP 20 d

RI 0.5l

RT 5g

RH 10 A~l

Sample 10 j

DEPCH20 114.5 d

2) Place the sample in a thermal cycler and incubated at the following settings:

(1) 25 -C 10min

(2) 42 -C 59 min

(3) 95 OC 5min

(4) so -C5min

3) After the processes from these (l)-{4) are completed, store the sample at -20 0C.

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7.6.2.3 PCR (polymerase chain reaction)

1) Add 5 ml of PCR mixture to the RT sample

PCR mixture:

MgCl2 1 l

l0OX PCRBuffer 2 d

ddH2O 16.4 ~

primer AS 0.25 l

primer S 0.25 ~i

Total 19.9 Wi per sample

2) Place the sample in the thermal cycler and add a drop of mineral oil.

3) Program the thermal cycler, for 20 -40 cycles as required, for gene amplification

The thermal cycler was programmed as follows:

9400C 45 sec

6000C 45 sec

72 0C 2min

4) After amplification, extract 10 p1 from the sample and add 1 p~l of Loading Buffer to the

fr-agments.

5) The PCR product is resolved in 110 g of 2 % agarose gel containing 10 0d of ethidium

bromide.

6) Electrophoreses at 100 volts for 1 hour.

7) The gel is illuminated from underneath with LTV light and photographed with Polaroid film.

8) The bands from the Polaroid photograph are scanned and the density of the bands is measured

using NMH image.

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M BO08T I

9) The ratio of the densities of the target genes to J3-actin genes as a control (internal standard).

7.6.3 TLMP2 measurements

The results of the measurements of TIMP2 in association with gene expression that is related to

fibrosis are shown in Figure 7.

1.50 ElD Control group

UTOFIX-S instlate group

_0 1.00

0.5

0.00

After 3 days After 6 months

Figure 7 Gene expression of TIMP in the lungs of rats at 3 days and 6 months after instillation.

-~Figure 7 shows that no differences were found in gene expression of TIMP in the instilled group

and the control group at 3 days and 6 months after instillation.

7.7 Hlistopathological examination

7.7.1 Lung tissue preparation

The lungs of 5 rats per group were sacrificed at 3 or 6 months after instillation of TOFDC-S and

examined bistopathologically. The lung was fully inflated by intratracheal infusion of 4%

parafonnaldehyde and fixed. After fixation, the left lung was sectioned along the sagittal plane to

include hilar surface so as to make the cut surfaces largest The lung sections were then embedded

in paraffin. Three pm-thick sections were then stained with hematoxylin and examined

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30 8 r

hitopathologically.

7.7.2 Results of bistopathological examination

7.7.2.1 Results of examination at 3 days after instillation

In the TOFIX-X instillated group, the degree of inflammation was only minimal and no significant

infiltration of inflammatory cells was observed. No specific aggregation of particles was observed

in the lung fields and lymph nodes, whereas 2 cases of spontaneous alveolitis were observed in the

control group. Micrographis 2 -4 represent the examples of bistopathological micrographs.

Oz

Micrograph 2 Alveolitis observed in the control group (3days after instillation)

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MBOST1

'A.

W

17N,

-

r

Mcrograph 3 TOFIX-S instillated group (3 days after instillation)

4 :

n -7

r

q

"A:' r:VK

Y e5i

14

wtw

r

7

Micrograph 4 Control group (3 days after instillation)

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7.7.2.2 Results of examination at 6 months after instillation

I the TOFDC-S instillated group, the degree of inflammation was only minimal and little changes

were observed compared with the control group. No findings including alveolitis were observed in

both groups.

Micrographs 5 and 6 represent the examples of bistopathological nmicrographs. The micrograph of

['Ti case is represented for reference.

4%4

-. 4K.- 

OK, YI ~ 4,, "M

-A. -

-, 1

- V

xA,

Micrograph TQJXSontolla group (6 months after instillation)

~~j~flPag 49 SA

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MIB 0O8T

Micrograph 7 PTI instillated group (6 months after instillation)

7.7.2.3 Inflammation point counting (degree of inflammation)

In the conventional histopathological analysis, the presence or absence of findings of tumors and

fibrosis and qualitative description of morphologic changes have been of major description. Certain

time course is required to progress to pulmonary fibrosis and tumors, however, changes in the

degree of inflammation in lung tissue in the process Deeds to be assessed quantitatively. In this

study, an attempt was made to analyze changes in the inflammation areas semiquantitatively under

an optical microscope.

1) Measurement method

Six images per specimen were randomly captured digitally under an optical microscope of 100

magnifications. To capture mainly lung fields, blood vessels and large bronchi were excluded. By

placing a 300-point grid over each image on the PC screen, the inflammatory areas in each image

were examined using the point counting method (1'CM. Six fields per section were examined-,

1,800 points in total, an average pulmonary area of 200 mm2 of cut surfaces on the preparations and

the total area of the images of 3.9 mm2 that was 2 % of the cut surface of the lung surface area were

examined. Excluding blood vessels and large bronchi, the point on the inflammatory areas

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including alveolar walls, alveolar ducts, terminal bronchioles and airspaces. Excluding airspaoes,

the percentage of inflammatory areas in the pulmonary parenchyma was calculated and group mean

was obtained.

2) Results of the inflammation degree evaluation

The degrees of inflammation were evaluated and the results are shown in Figure 8.

No significant differences were found both at 3 days and 6 months after instillation compared to the

control group. The percentage of the inflammation resulted from TOFDC-S instillation was

averaged less than 1I% only a minimal or no inflammation was observed.

0.05

0.04

0.03 - - 7

TOMI

0.02

0.01

0

3d 6

Figure 8 Degrees of inflammation

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Chapter 8 Discussion

8.1 Rat growth curves

No differences were found in the growth curves of the body weight between two groups.

This suggests that exposure to TQF]X-S did not have an effect on the growth of the rats.

8.2 Wet organ weights of the rats at each time point of sacrifices

No significant differences were found in the weight of any of the lung, liver, kidney or spleen of the

rats between two groups.

8.3 Biopersistence of TOFIX-S

The biological half time (Ti,2) of the lung burden of TOFDC-S calculated by using the measured

lung burdens was 2.2 months.

T1t2 for PT1I (potassium octatinate wisker) that has been stated earlier for reference is 12 months.

It is postulated that the longer retention in the lung increases the length of time of contacting the

body, and induces various kinds of biological reactions at higher degrees. It has been assumed that

the clearance half time of asbestos is on the scale of years, its hazardous potential is much greater.

Since the biological half time of 2.2 months obtained in this study is similar to that of particulate

materials obtained in our laboratory, TOFIX-S are predicted to have little biological effect.

8.4 8-hydroxydeoxyguanosine

No significant formation of 8-hydroxydeoxyguanosine was observed compared to the control

group.

Significant increase in the formation of 8-hydroxydeoxyguanosine occurred as a major product of

oxidative damage caused by active oxygen species, indicating the substance's higher potency of

hazard. Therefore, the substance can be considered to have higher hazardous potential.

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In this study, however, no production of 8-hydroxydeoxyguanosine was observed following

TOFIX-S instillation, it can be predicted that significant pulmonary injury and carcinogenesis

induced by active oxygen species will not occur.

8.5 TIMP-2

No significant differences in gene expression of TIMP-2 between two groups were found. From

this, it can be predicted that TOFIX-S has little effect on inflammation and fibrosis

-These results are consistent with the histopathological examination in this study.

8.6 Hfistopathological changes

Hfistopathological examination showed no significant differences between two groups and the

biological half time of TOFDC-S was 2.2 months which was quite short. These results are consistent

with the postulate that TOFIX-S have little biological effect.

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Chapter 9 Conclusion

Particles constitute most of TOFIX-S, but it also contains fibrous particles as well. When there had

been concern about potential health effects of asbestos, fibers longer than 20 pm were of potential

health concern. No such fiber lengths were found for TOFDC-S. -

The assessment of the biopersistence or durability in the lung is known to be an index to predict the

level of biological effects of fibrous materials. The biological half time or biopersistence of

TOFIX-S was 2.2 months that is consistent with that of particulate materials obtained in our

laboratory which have little biological effect.

The results of the measurements of TLMP2 in association with gene expression that are indices for

inflammation and fibrogenesis were not those predictive of fibrogenesis. This was consistent with

the pathologic features.

For 8-hydroxydeoxyguanosine, no formation was observed. No significant changes were observed

in the histopathological examination of the lungs.

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