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June 8, 2007



## Research Report


# Biological Effects of TOFIX-S Using IntratrachealInstillation

Professor Isamu ak Institute of Industrial Ecological Sciences University of Occupational and Environmental Health, Japan Page 19MB08TI 

## 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 ofTOFIX-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 ofthe organs at each time point of sacrifices 7.3 Quantification of lung burdens ofTQFIX-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 Page 20MBO08T
7.7 Histopathological examination Chapter 8 Discussion 8.1 Rat growth curves 8.2 .We-t organ weights ofthe 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

8Figures 3 Tables 7 Micrographis

Page 21MB08 T 1
Chapter 1 Research Project and Research Team Research Project Biological Effects ofTOFIX-S UsingIntratracheal Instillation Research Team Lead investigator: Professor Isamu Tanaka, Dr. Eng., Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated from the Department ofEngineering, Kyushu University in 1967) Researchers involved in the project:

- Professor Yasuo Morimoto, MD, DMS, Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated fromKagoshima University School ofMedicine in 1986) Associate Professor Toshihiko Myojo, Dr. Eng., Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated from Department ofEngineering, Kanazawa University in 1977) Associate ProfessorAkira Ogami, MI), DMS,

______________ Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated from University ofOccupational and Environmental Health Graduate School Page 220BOTl
ofMedicine in 1997) Assistant Professor Takako Oyabu,DM3, 

# 7 rt Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated from the Department ofScience, University ofKyushu in 1983)

Page 23MBOST1 Chapter 2 Research Objectives The aim ofthis study is to assess the potential biological hazards ofTOFDC-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 tofibrogenesis 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 ofthe -diseases vary greatly according to the dust type. For pulmonary fibrosis (pneumoconiosis) and canoers in the lungs, -itis 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 ofvarious 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 oflung clearance ofTOFIX-S. Histopathological examination and cytokines which are responsible for fibrotic and carcinogenic changes in the lung are examined to assessthe biological effects ofTOFDC-S.

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### Chapter 3 Research Design and Methods The biopersistence ofchemicals that are introduced into the lung via airways is vital to assess their health effects orbiological effects inhumans. The fate ofchemical substances in the lung depends largely ontheir physiochemical characteristics, dimension and especially biopersistance in the lung, over time, that is involved directly with the length oftime required for the initiation ofbiological responses and the effects ofsuch responses. The significance ofthis understanding has begun to be appreciated in recent years and, therefore, biopersistence offibrous dustwhich contributes especially to fibrogenesis and carcinogenesis in the lung have being investigated asthemost 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 inthe lungs. On this basis, we planned and conducted an experiment according to the following procedures to obtain our objective: I. Identify the geometric shapes ofTOFIX-S by electron microscopy 2. Conduct an animal experiment in whichTOFDC-S is instilled intratracheally into rats 3. Decide which methodto use for quantification ofTOFIX-S entered inthe animal lung 4. QuantifyTOFIX-S retained in the lungs to determine the rate oflung clearance by means of serial sacrifices ofa givennumber ofrats at certain time points 5. Measure the amount ofcytokine which is deeply involved in fibrotic or carcinogenic changes inthe lungs; 6. H1istopathological examinations ofthe lungs

Page 25MBOST1 Chapter 4 TOFIX-S Particle Characteristics 4.1 Contents offibrous materials Filters containing TOFIX-S and deposited platinum on them were prepared for scanning -electron microscopy assessment ofTOFTX-S lengths and diameters; and, subsequently digital micrographs were made (Micrograph 1).

Micrograph 1 A scanning electron micrograph ofTOFIX-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 ofview ofthe scanning electron microscope were counted. Thenumber of fibrous particles and non-fibrous particles quantified by the scanning electron microscopy were 124 and 1,136, respectively. This indicates that the number offibrous particles accounts for 9.8 % oftotal number ofparticles counted. Since a product that contains asbestos exceeding 0.1 %by weight which asbestos bears tothe whole product is defined as an asbestos product according to the Ordinance on the Prevention ofHazards due to Asbestos, TOFJX-S can be defined as an fibrous product, therefore, wethough thatit is relevant to assess the potential hazards ofTOFDC-S. Page 26MBO8T1 4.2 Fiber diameters and lengths Diameter and length are the basic determinants offibrous materials. TOFIX-S was collected Onthe 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 inthe Figures 1and 2.
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- 7 U 10 UUS90U 5U 7010 ~Pg 207MB08TI

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 imand 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 ofthe fibers were found to be less than 0.8 in.in diameter but were shorterthan20 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 longerthan 20pm were found forTOFIX-S.

Page 28MB08T 1 Chapter 5 TheRecovery ofTQFLX-S from theAnimal 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 inthe animal tissue, to determine lungburdens. The samples to obtain recovery rates were prepared by first taking a precise amount ofTOFDC-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 -givenamount ofTOFIX-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 ofeither 0, 0.05, 0.1, 0.2,0.5, 1.0 or 2.0 mg was added to each Ig ofhomogenized liver andeach 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 inFigure 3,to obtain recovery rates. The amount ofTQFDC-S in each sample which -contained50.1 %Ti was calculated by converting measured Ti concentrations. The results are shown inTable 1. The results showed that almost 100% ofTOFIX-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 ofTQFIX-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 conditions

# C250W 3min 500W 20mm 

# J

Preare

tocontan voumeof50m
Quantify Ti amount (ICP-AES analysis) * (1O0ml+40g) H 2SO 4+(NH4)SO4 Figure 3 Procedures forthe quantification ofTOFLX-S lungburdens Page 30MBO8T1
Table 1 The results ofrecovery 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 0.4521 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 *Excludedfromthe datadue to an error inmeasurment

page 3 1MB08 T 1 Chapter 6 Intratracheal Instillation 6.1 Instillation study Potential hazards ofchemical substances in human differ significantly with the routes ofexposure (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 oftest materials in the animal lung. Agiven amount oftestparticles suspended ina solution is injected by syringe through the cannula into the trachea, delivering the test particles directly into the lungs. Regarding the advantages and disadvantages ofthemethod, intratracheal instillation administers an accurate dosage ofmaterials but it has the disadvantages that large amount ofdose 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 ofintroduction into body is an unnatural route ofexposure in human exposure, but the method does provide useful information on how the test particles behave in the lungs. The main theme ofthis study isto estimate the biological effects ofTOFIX-S. For this purpose, we conducted an intratracheal instillation experiment to examine the biopersistence ofTOFLX-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 ofrats Page 32MBO8T1 Seventy rats were randomly divided intotwo similarly sized groups: Intratracheal instillation group: TQFIX-S exposing group Control group: Physiological saline exposing group 6.2.3 Administratiion ofTOFIX-S TOFIX-S was suspended in physiological saline at the rate of2 mig per 0.4 ml and administered to 35 rats by a single intratracheal instillation intothe under ether anesthesia. Rats in thecontrol group were given only the 0.4 ml ofphysiological 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 ratsper group were sacrificed.

- 1 month after instillation 5 ratsper group were sacrificed.

-3 months after instillation 5 rats pergroup were sacrificed.

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

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### M480o87,



### Chapter 7 Results oftheIntratracheal Instillation 7.1 Rat growth curves The growth curves oftherats 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 inrat 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 tobe noted. 7.2 Wet weights ofthe organs at eachtime pointof sacrifices The results ofthe measurement ofthe wet weights ofrat lung, liver, kidney and spleen at each time point ofsaciffices are shown in Table 2.

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Table 2 Wet weights (g)ofrat lung, liver,kidney and spleen at each time point ofsacrifices. 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 1week 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 1month Controlgroup 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-Shwd&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 inthe two groups. 7.3 Quantification oflung burdens ofTOFIX-S 1) Method for quantification ofTOFIX-S in the lungs Five rats in each group were sacrificed at given time points to quantify the amount ofTOFIX-S retained in the lungs following the procedures described in Figure 3. The amount ofTOF1X-S in each sample which contained 50.1 %Ti was calculated by converting measured Ti amounts.

Page 35MBO8T 1
2) Lungburden ofTOFIX-S The results ofmeasurements are shown in Table 3. Table 3 Lung burden ofTOFJX-S at each timepoint ofsacrifices Lung burden ofTOFDC-S (mg) 3days after instillation 1.21 ±0.28 1week after instillation 0.73 ±0.22 1month 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 ofthe control groups were found to be extremely low (less than 1pg) when compared with to those in rat lungs ofthe TOFIX-S instillated group, those concentrations were regarded as zero in quantifying the amount ofTOFIX-S inthelungs. Itwas found that the lung burden ofTOFIX-S decreased with time after instillation. The dose ofTOFD(-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 ofthem 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 ofapproximately 3X 1 08 alveoli oftiny air sacs with a surface are of about 200 pm, having a total surface area ofabout 100 square nilimeters. Pulmonary alveoli are surrounded on the outside with fine blood vessels. It is in the alveoli in the lungs thatgas 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 Page 36MB08T1 the lymphatic system. The values shown in Table 3 may indicate that TOFDC-S deposited in the alveolar region was cleared from thelungs by these clearance mechanisms. 7.4 Biopersistence ofTQFIX-S from the lungs (biological halftime) Relationship between the lung burden ofTOFIX-S at each given time point after instillation were plotted on semi-logarithmic graph to estimate the rate ofTOFIX-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 halftime: 12 months IPTZS

Biological halftime: 2.2 months 0.1 -I I __ - I 0 1 2 3 4 5 6 Time points after instillation (month) Figure 5 Changes oflung burden ofTOFIX-S and PTlI with time The results obtained in our laboratory for the changes oflung burden ofpotassium octatitanate whisker (PT 1)with time are indicated in Figure 5in 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 thatcan be obtained by lungburdens ofTOFIX-S measured at three days, 1 Page 37MB08TI
week, 1 month, 3 months and 6 months after instillation (Co). This can be summarized by the formula: dC/dt =-kC(1 Initialconditio: 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 ofthe experiment Then, the following equation isobtained when Equation (1)is solved usingthe 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 ofparticles is cleared from the lung. The T 1t2 is given by using the following Equation (4): Ti2= 0.693/k (4) The biological halftime 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 ofthe line shown in Figure 3.T1/ 2 forPTl (potassium octatinate wisker) is 12 months. 7.5 Formation of 8-hydroxydeoxyguanosine in lungtissue 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 Page 38MBO8T1 reported. 7.5.1 Measurement method 1) Flush a 1.5 ml centrifuge tube with nitrogen, 2) Put the lung tissue inthe tube and add 1ml Lysis solution, 3) Hold the tube on the ice forhomogenization, 4) Centrifuge at 10,000 gfor 20 seconds, 5) Discard the supernatant and add ImlLysis solution aan 6) Centrifuge at 

### 10,000 gfor 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 gfor 10 minutes, 11) Discard the supernatant, 12) Add0.3m1 NaI solution, 13) Add 0.5m1 isopropyl alcohol, 14) Centrifuge at 10,000 gfor 10 minutes, Page 39M B08T 1
15) Discard the supernatant, 16) Add 1ml A (wash fluid) and washby centrifugation, 17) Add 1ml B (wash fluid) and wash by centrifugation, 18) Drying 19) Add 0.1 imI1mMIEDTA, 1J4 I2M of sodium acetate buffer solution, 4 0d of5mg/mi nuclease P1, and 2 W.ofphosphoric acid; and incubate at 37 00 for 30 minutes, 20) Perform ion-exchange resin and centrifuge, 21) Transfer the solution to atube 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/dGratio to quantify 8OHdG. With the 8OHdG/dG ratio, the amount of8OHdG canbe determined. 7.5.2 Measurements of 8-hydroxydeoxyguanosine The levels of 8-hydroxydeoxyguanosine measured in the lungs ofrats 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 3days after instillaton 6months afterinstilton Figure 6 The levels of8OHdG in

### the lungs ofrat sacrifices at 3days and 6 

### months after instillation No significant differences were found between the instilled group and the control group for the levels of8OHdGboth at 3days and 6months after instillation. 7.6 Expression ofgene related to inflammation and fibrosis inlungtissue 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 ofcollagen. 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 ofcollagen. 7.6.1.1 Excessive production ofcollagen Collagen in lung tissue is primarily ofType I collagen and it has been postulated that the extensive deposition ofType Icollagen induces fibrosis. The occurrence ofexcessive production ofcollagen couldbe determined from examination ofwhether gene expression ofType I collagen increased. Page 417.6.1.2 Regulation ofcollagen degradation ?l0T Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (MMPs) are matrix-degrading enzymes. Deposition ofcollagen 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 ofTIPs than ofMM~s may contribute tothe downregulation ofdegradation resulting in the deposition ofcollagen Alate stage or fibrotic stage in the process of fibrosis, the gene expression of TRAPi is elevated. Conversely, when the level ofMIMP activation is higher than that of TIMvIP, the degradation of collagen is facilitated, which in turn results in lesser deposition ofcollagen. This isseen in anearly stage or lung injury and inflammation in the process offibrosis and also seen in pulmonary lung masses that canbe attributedto cigarette smoking. The mechanism of fibrosis caused whether by increased production of collagen or by regulated degradation ofcollagen can be elucidation bymeans ofquantification and analysis ofthe results. It seems a reasonable procedure to first conduct a screening for expression ofthese genes, then, if significant changes are found in any ofthese three genes, expression ofthe other two genes are further examined. We decided to first measure the activation ofTIMP and, ifsignificant differences in the levels of'lIM were found compared to the control group, then further measure the levels of 'TypeI collagen andMMP.R 7.6.2 Measurement method 7.6.2.1 RNA extraction procedures Lung tissue was homogenized with 1.2 ml ofSolution 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 ofPhenol DEPC Page 42MBO08T I
1120 and 120 p1ofChioroform-Isoamyl alcohol (49:1) to thehomogenized solution, 2) The mixture is agitated for 10 seconds and held onthe ice. 3) Centrifugate at 15,000 rpm for 20minutes at400, 4) Inject the supernatant in the PHASE LOCK GEL(PLG) tube and centrifugate at 8,000 rpm for 5minutes, 5) Transfer the resulting supernatant to a new centrifuge tube, mix with 1.2 ml ofisopropanol and hold at -20'Cfor 15 minutes, 6) Centrifugate at 15,000 rpm for 20 minutes at 4*C, 7) Discard the supernatant and add 360 p.1 ofSolution D to the pellet, 8) Add 360 0. ofisopropanol 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 of75% ethanol and centrifuge at 15,000rpm for 10 minutes at4*C, 12) Discard the supernatant and centrifuge for 5minutes while sucking, 13) Sufficiently dry the RNA pellet andadd TE20-50 W.to dissolve the pellet, 14) Adjustthe concentration to 2.2 pgg/pil by using spectrometer, After extracting the RNA, gene expression ofTRAvP was measuredby perform reverse-transcription polymerase chain reaction (RT PCR). Page 43MBO08T 1

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 eppendorftube, add a sample of 10 ~ of tissue (contains 22 gg ofRNA), and dilute the mixture with 114.5 RIofDEPC H20 to 200jI in total. RT-mixture: 5XRTbuffer 40 ~ d-NTP 20 d RI 0.5l RT 5g RH 10 A~l Sample 10 j DEPCH2 0 114.5 d 2) Place the sample ina thermal cycler and incubated at the following settings: (1) 25-C 10min (2) 42-C 59min (3) 95 OC 

### 5min (4) so -C5min 3) Afterthe processes from these (l)-{4) are completed, store the sample at -20 0C.

Page 44MD08TI

7.6.2.3 PCR (polymerase chain reaction) 1) Add 5ml ofPCR 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 persample 2) Place the sample in the thermal cycler and add a drop ofmineral oil. 3) Program the thermal cycler, for 20-40 cycles asrequired, for gene amplification The thermal cycler was programmed as follows: 9400C 45 sec 6000C 45 sec 720C 2min 4) After amplification, extract 10 p1 from the sample and add 1 p~l ofLoading 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 1hour. 7) The gelis illuminated from underneath with LTVlight and photographed withPolaroid film. 8) The bands from the Polaroid photograph are scanned and the density ofthe bands is measured using NMH image. Page 45MBO08T I
9) The ratio ofthe densities ofthe target genes to J3-actin genes as acontrol (internal standard). 7.6.3 TLMP2 measurements The results ofthe measurements ofTIMP2 in association with gene expression that is related to fibrosis are shown in Figure 7. 

### 1.50 

### ElD Controlgroup 

### UTOFIX-S instlate group _0 1.00

0.5

0.00 After 3days After 6 months Figure 7 Gene expression ofTIMP inthe lungs ofrats at 3days and6 months after instillation.

-~Figure 7 shows that no differences were found in gene expression ofTIMP in the instilled group and the control group at3 days and 6 months after instillation. 7.7 Hlistopathological examination 7.7.1 Lung tissue preparation The lungs of 5rats per group were sacrificed at 3 or 6 months after instillation ofTOFDC-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 Page 46

### 0 3 8

### r hitopathologically. 7.7.2 Results ofbistopathological examination 7.7.2.1 Results ofexamination at 3days after instillation In the TOFIX-X instillated group, the degree ofinflammation was only minimal and no significant infiltration of inflammatory cells was observed. No specific aggregation ofparticles was observed in the lung fields and lymph nodes, whereas 2 cases ofspontaneous alveolitis were observed in the control group. Micrographis 2-4 represent the examples ofbistopathological micrographs.

Oz

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

Page 47MBOST1

'A.

W

17N,

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

4 :

n -7 r q "A:'r:VK
Y e5i 14 

### wtw r 7 Micrograph 4 Control group(3 days after instillation) Page 487.7.2.2 Results ofexamination at 6months after instillation I the TOFDC-S instillated group, the degree ofinflammation 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 ofbistopathological 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 SAMIB 0O8T

Micrograph 7 PTI instillated group (6 months after instillation) 7.7.2.3 Inflammation point counting (degree ofinflammation) In the conventional histopathological analysis, the presence or absence of findings oftumors and fibrosis and qualitative description ofmorphologic changes have been ofmajor 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 inthe 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 of200 mm 2 ofcut surfaces onthepreparations and the total area ofthe images of3.9 mm 2 thatwas 2 %ofthe cut surface ofthe lung surface area were examined. Excluding blood vessels and large bronchi, the point on the inflammatory areas Page 50MB0 8TI
including alveolar walls, alveolar ducts, terminal bronchioles and airspaces. Excluding airspaoes, the percentage ofinflammatory areas in the pulmonary parenchyma was calculated and group mean was obtained. 2) Results ofthe inflammation degree evaluation The degrees ofinflammation were evaluated and the results are shown inFigure 8. No significant differences were found both at3 days and 6months after instillation compared tothe control group. The percentage of the inflammation resulted from TOFDC-S instillation was averaged less than 1I% only aminimal or noinflammation was observed.

0.05
0.04
0.03 - - 7 TOMI 0.02
0.01
0 3d 6 Figure 8 Degrees ofinflammation

Page 51Chapter 8 Discussion 8.1 Rat growth curves No differences were found in the growth curves ofthe body weight between two groups. This suggests that exposure to TQF]X-S didnot have an effect on the growth ofthe rats. 8.2 Wet organ weights oftherats ateach timepoint ofsacrifices No significant differences were found in the weight ofany ofthe lung, liver, kidney or spleen ofthe ratsbetween two groups. 8.3 Biopersistence ofTOFIX-S The biological half time (Ti,2) ofthe lung burden ofTOFDC-S calculated by using the measured lung burdens was 2.2 months. T

1t2forPT1I (potassium octatinate wisker) thathas been stated earlier for reference is 12 months. It is postulated that the longer retention in the lung increases the length oftime ofcontacting the body, and induces various kinds ofbiological reactions at higher degrees. It has been assumed that the clearance halftime ofasbestos is onthe scale ofyears, its hazardous potential is much greater. Since the biological halftime of 2.2 months obtained inthis study is similar to that ofparticulate materials obtained in ourlaboratory, TOFIX-S are predicted tohave 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. Page 52MBO8Tl

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 ofTIMP-2 between two groups were found. From this, it can be predicted thatTOFIX-S has little effect oninflammation and fibrosis

-These results are consistent withthe histopathological examination in this study.

8.6 Hfistopathological changes Hfistopathological examination showed no significant differences between two groups and the biological halftime ofTOFDC-S was 2.2 months which was quite short. These results are consistent withthe postulate that TOFIX-S have little biological effect.

Page 53Chapter 9 Conclusion Particles constitute most ofTOFIX-S, but it also contains fibrous particles as well. When there had been concern about potential health effects ofasbestos, fibers longer than 20 pm were ofpotential health concern. No such fiber lengths were found forTOFDC-S.

- The assessment ofthebiopersistence or durability in the lung is known to be an index topredict 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 littlebiological effect. The results ofthe measurements ofTLMP2 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 ofthe lungs.

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