臺灣博碩士論文加值系統

在防治含氯有機溶劑污染水源與土壤的研究課題上，應用細胞培養技術，以明瞭並減低人類公共衛生所承受的危險，將是個有力工具。就三氯乙烯與四氯乙烯而言，已經是一個普遍公認的工業化所造成的環境污染問題，因為此化學物質在工業上所使用的範圍非常地廣泛。本篇研究的目標在於利用中國倉鼠細胞，印證動物活體於三氯乙烯及四氯乙烯在不同濃度，對細胞的生長抑制和生化代謝毒性的影響。
就中國倉鼠卵巢細胞對於三氯乙烯與四氯乙烯的毒性確認，是基於將細胞接種於活體外的揮發暴露系統中。在特製60 mm 培養皿中間區域部分，另外貼黏著一個25-mm 大小的玻璃培養皿，以提供加入揮發性藥劑所需要的區域。個別處理三氯乙烯或四氯乙烯的劑量分別是三氯乙烯20-80 ul ，四氯乙烯5-20 ul ；其中利用藥劑揮發後I變成氣體，再溶到培養液中。進而研究其影響細胞的生理與生化之反應。得到中國倉鼠卵巢細胞對三氯及四氯乙烯的毒性影響有：細胞的生長抑制、細胞內的酵素活性、細胞內GSH 的濃度等。其中明顯隨著添藥量增加而明顯增加的是：生長抑制現象與微小核增加頻率等。此外也發現在低劑量處理時 (三氯乙烯5-20 ul ，四氯乙烯1-5 ul) GSH 含量反而會隨之增加。增加量約為1.5 倍正常量。但當添藥量超過之後GSH 則是快速的下降。除此之外在GST 的活性方面，活性強度隨著處理藥劑濃度的增加，而有明顯的增加，上升的倍數約為三倍值。比較GSH 含量不同之後發現，隨著藥劑濃度增加，而並沒有顯著地改變細胞的生長抑制現象。在三氯與四氯乙烯對中國倉鼠卵巢細胞毒性上，GSH-conjugate 可能是一個中間反應；然而GSH-dependent 的活化作用在中國倉鼠卵巢細胞中，對三氯與四氯乙烯的biotransformation 上扮演了一個次要的解毒角色。

The development and validation of cell culture techniques for
estimating chlorine contained organic solvents in contaminated soil and water can facilitate the overall understanding of anamal health risk. Currently, trichloroethylene (TCE) and tetrachloroethylene (PCE) are the major environmental contaminant and an occupational concern issues because of their widespread industrial use. The objective of this study was to use the cell culture techniques (Chinese hamster ovary cells, CHO-K1) to determine the toxicity of the toxicity of trichloroethylene (TCE) and tetrachloroethylene (PCE) on the CHO-K1. The CHO-K1 cells were grown in a 60-mm petri dish with a 25-mm glass dish glued in the central area. The TCE (20-80 ul) or PCE (5-20 ul) was added to the central glass well, and let it to evaporate and dissolve in the surrounding medium in
which cells were growing. Treatment of CHO-K1 cells with either TCE or PCE resulted in a dose and exposure inhibition of cell growth. A statistically significant increase in the frequency of MN (micronuclei test) was also observed in either TCE or PCE-treated CHO-K1 cells. Low dose of TCE (5-20 ul) or (1-5 ul) treatments showed significantly enhancement the intracellular glutathione (GSH) levels. However, the level of GSH rapidly decreased in the presence of higher dose of TCE (40-80 ul) or PCE (10-20 ul). Furthermore, the GST activity was enhanced by TCE- or PCE- treatment. Depletion of cellular GSH showed
no effect on the sensitivity of CHO-K1 cells to TCE- or PCE- treatments. The GSH-conjugate has been proposed as a reactive intermediate, which is account for the toxicity of TCE and PCE, however the GSH-dependent activation play a minor role in the biotransformation of TCE and PCE in CHO-K1cells.

目錄
壹、 中文摘要… … … … … .… … … .. … … … … . … ..IV
貳、 英文摘要… … … … ….. .. … … .. … … … … . … ..VI
參、 緒論… … … … … … …… … ... … … . … … … … . 1
肆、 材料與方法.. … … … …. … .. … . … … . … … … .11
伍、 結果… … … … … … … … … … … … … … … … ...19
陸、 討論… … … … … … … .. … … … … … … … … ...23
柒、 圖表… … … … … … … … … … … … … … … … ...28
圖一、有機溶劑對細胞生長抑制的情形… …. … … … … … … .28
圖二、有機溶劑處理後24 小時生長觀察… …… … … … … … . 29
圖三、細胞毒性的分析… … … … … .. . … … .. … … … . 30
圖四、細胞內Glutathione 的分析… … … … … … … … … … 31
圖五、經BSO 前處理後再暴露在有機溶劑的結果(一) … …..... ..32
圖六、經BSO 前處理後再暴露在有機溶劑的結果(二) … …..... ..33
圖七、細胞Glutathione S Transferase (GST) activity 分析…...34
圖八、細胞內Catalase 的分析… … … … … … … … … … .35
圖九、微核的觀察… … … . … … … … . … … … … … … 36
圖十、酸鹼的變化現象… … . … … … … …. … … … … … .37
捌、 參考文獻… … … … … … .. … … … … … … … . … 38
玖、 附錄… … … … … … … .. … . … …… … … .. … ..46

參考文獻 (References)
註1 ：中央日報，1998 年6 月01 日
http:// www.cdn.com.tw/daily/1998/06/01/text/870601dh.htm
註2 ：中央日報，1998 年6 月02 日
http://www.cdn.com.tw/daily/1998/06/02/text/870602dm.htm
註3 ：中央日報，1998 年8 月13 日
http://www.cdn.com.tw/daily/1998/08/13/text/870813da.htm
註4 ：中央日報，1998 年6 月22 日
http://www.cdn.com.tw/daily/1998/06/22/text/870622dh.htm
註5 ：整治進度，網頁
http://www.epa.gov.tw/news/en880208.htm
柯青水,1989 ，“化學化工大辭典”,正文書局出版
Aebi, H. ( 1984 ) Catalase in vitro. Methods Enzymol., 105, 121-126.
Axelson O, Andersson K, Hogstedt C, Holmberg B, Molina G, de
Verdier A ( 1978 ) A cohort study on trichloroethylene exposure and
cancer mortality. J Occup Med., 20, 194-6.
Axelson, O ( 1978 ) Exchange of oxime functions: a useful reaction in
GC-ms analysis of steroids. Anal Biochem., 86, 133-41.Baccarelli A ( 1999 ) Occupational agents and endocrine function: an
update of the experimental and human evidence. Med Lav., 90, 650-
70.
Boice JD Jr, Marano DE, Fryzek JP, Sadler CJ, McLaughlin JK
( 1999 ) Mortality among aircraft manufacturing workers. Occup
Environ Med., 56, 581-97.
Bove FJ, Fulcomer MC, Klotz JB, Esmart J, Dufficy EM, Savrin JE
( 1995 ) Public drinking water contamination and birth outcomes.
Am J Epidemiol., 141, 850-62.
Bradford, M. M. ( 1976 ) A rapid and sensitive method for the
quantitation of microgram quantities utilizing the principle of
protein-dye binding. Anal. Biochem.,72, 248-254.
Bruning T, Bolt HM ( 2000 ) Renal toxicity and carcinogenicity of
trichloroethylene: key results, mechanisms, and controversies.Crit
Rev Toxicol., 30, 253-85.
Bruning T, Mann H, Melzer H, Sundberg AG, Bolt HM ( 1999 )
Pathological excretion patterns of urinary proteins in renal cell
cancer patients exposed to trichloroethylene. Occup Med (Lond)., 49,
299-305.
Bruning T, Sundberg AG, Birner G, Lammert M, Bolt HM,
Appelkvist EL, Nilsson R, Dallner G ( 1999 ) Glutathione
transferase alpha as a marker for tubular damage after
trichloroethylene exposure. Arch Toxicol., 73, 246-54.
Cohn, V. H., and Lyle, J. ( 1966 ) A fluorometric assay for glutathione.
Anal. Biochem.,14, 434-440.
Cummings BS, Parker JC, Lash LH ( 2000, a ) Role of cytochrome
P450 and glutathione S-transferase alpha in the metabolism and
cytotoxicity of trichloroethylene in rat kidney. Biochem Pharmacol.,
59, 531-43.
Cummings BS, Lash LH ( 2000,b ) Metabolism and toxicity of
trichloroethylene and S-( 1,2-dichlorovinyl )-L-cysteine in freshly
isolated human proximal tubular cells. Toxicol Sci., 53, 458-66.
D. Alan Warren, Thomas G. Reigle, Srinivasa Muralidhara, Cham E.
Dallas ( 1995 ) Schedule-Controlled Operant Behavior of Rats
following oral administration of perchloroethylene: time course and
relationship to blood and brain solvent levels. Journal of Toxicology
and Environmental Health., 47, 345— 362.
Elcombe CR ( 1985 ) Species differences in carcinogenicity and
peroxisome proliferation due to trichloroethylene: a biochemical
human hazard assessment. Arch Toxicol Suppl., 8, 6-17.
Elcombe CR, Rose MS, Pratt IS. ( 1985 ) Biochemical, histological,
and ultrastructural changes in rat and mouse liver following
theadministration of trichloroethylene: possible relevance to species
differences inhepatocarcinogenicity. Toxicol Appl Pharmacol.,79,
365-76.Forkert PG, Birch DW ( 1989 ) Pulmonary toxicity of trichloroethylene
in mice. Covalent binding and morphological manifestations. Drug
Metab Dispos., 17, 106-13.
Forkert PG, Sylvestre PL, Poland JS ( 1985 ) Lung injury induced by
trichloroethylene. Toxicology., 35, 143-60.
Giovannini L, Guglielmi G, Casini T, Bertelli A, Galmozzi E, Bertelli
AA ( 1992 ) Effect of ethanol chronic use on hepatotoxicity in rats
exposed to tetrachloroethylene. Int J Tissue React., 14, 281-5.
Goel SK, Rao GS, Pandya KP, Shanker R. ( 1992 ) Trichloroethylene
toxicity in mice: a biochemical, hematological and pathological
assessment. Indian J Exp Biol., 30, 402-6.
Habig, W. H., and Jakoby, W. B. ( 1981 ) Assay for differentiation of
glutathione S-trans-ferases. Methods Enzymol., 77, 398-405.
Habig, W. H., Pabst, M. J., and Jakoby, W. B. ( 1974 ) Gluthione S-transferase,
the first enzymatic step in mercapturic acid formation. J. Biol.
Chem., 249, 7130-7139.
Joel LA ( 1995 ) Alternative solutions to health problems. Am J Nurs., 95,
7
Kaneko T, Wang PY, Sato A, Related Articles ( 1997 ) Assessment of
the health effects of trichloroethylene. Ind Health., 35, 301-24.
Review.
Kumar P, Prasad AK, Dutta KK ( 2000 ) Steroidogenic alterations in
testes and sera of rats exposed to trichloroethylene ( TCE ) by
inhalation. Hum Exp Toxicol., 19, 117-21
Lash LH, Fisher JW, Lipscomb JC, Parker JC. ( 2000 ) Metabolism of
trichloroethylene. Environ Health Perspect.,108 Suppl 2, 177-200.
Lash TL, Green LC ( 1993 ) Blink reflex measurement of effects of
trichloroethylene exposure on the trigeminal nerve. Muscle Nerve.,
16, 217-20.
Lash LH, Qian W ( 1998 ) Glutathione conjugation of perchloroethylene
in rats and mice in vitro: sex-, species-, and tissue-dependent
differences. Toxicol Appl Pharmacol., 150, 49-57.
Lawrence H. Lash, Wei Qian, David A. Putt, Ketan Desai, Danan A.
Elfarra, Anna Rita Sicuri, and Jean C. Parker ( 1997 ) Glutathione
conjugation of perchloroethylene in rats and mice in Vitro:sex-, species-,
and tissue-dependent differences. Toxicol. Appl. Pharmacol., 150, 49—57.
Leandri M, Schizzi R, Scielzo C, Favale E( 1995 ) Electrophysiological
evidence of trigeminal root damage after trichloroethylene exposure.
Muscle Nerve.,18, 467-8.
Lee KM, Muralidhara S, Schnellmann RG, Bruckner JV ( 2000 )
Contribution of direct solvent injury to the dose-dependent kinetics
of trichloroethylene: portal vein administration to rats. Toxicol Appl
Pharmacol., 164, 46-54.
Lee TC, Ko JL, Jan KY ( 1989 ) Differential cytotoxicity of sodium
arsenite in human fibroblasts and Chinese hamster ovary cells.
Toxicology 56, 289-99.
Lochmann ER, Ehrlich W, Mangir M ( 1984 ) The effect of
trichloroethylene and acrylonitrile on RNA and ribosome synthesis
and ribosome content in Saccharomyces cells. Ecotoxicol Environ
Saf., 8, 162-6.
Mattsson JL, Albee RR, Yano BL, Bradley G, Spencer PJ ( 1996 )
Neurotoxicologic examination of rats exposed to 1,1,2,2-
tetrachloroethylene ( perchloroethylene ) vapor for 13 weeks.
Neurotoxicol Teratol., 20, 83-98.
.
Moser VC, Cheek BM, MacPhail RC ( 1995 ) A multidisciplinary
approach to toxicological screening: III. Neurobehavioral toxicity. J
Toxicol Environ Health 45, 173-210.
Muller Popp W, G, Baltes-Schmitz B, Wehner B, Vahrenholz C,
Schmieding W, Benninghoff M, Norpoth K ( 1992 )
Concentrations of tetrachloroethene in blood and trichloroacetic acid
in urine in workers and neighbours of dry-cleaning shops. Int Arch
Occup Environ Health., 63, 393-5.
Nakai JS, Stathopulos PB, Campbell GL, Chu I, Li-Muller A, Aucoin
R ( 2000 ) Penetration of chloroform, trichloroethylene, and
tetrachloroethylene through human skin. J Toxicol Environ Health.,
58, 157-70.
Narotsky MG, Weller EA, Chinchilli VM, Kavlock RJ ( 1995 )
arsenite in human fibroblasts and Chinese hamster ovary cells.
Toxicology 56, 289-99.
Lochmann ER, Ehrlich W, Mangir M ( 1984 ) The effect of
trichloroethylene and acrylonitrile on RNA and ribosome synthesis
and ribosome content in Saccharomyces cells. Ecotoxicol Environ
Saf., 8, 162-6.
Mattsson JL, Albee RR, Yano BL, Bradley G, Spencer PJ ( 1996 )
Neurotoxicologic examination of rats exposed to 1,1,2,2-
tetrachloroethylene ( perchloroethylene ) vapor for 13 weeks.
Neurotoxicol Teratol., 20, 83-98.
.
Moser VC, Cheek BM, MacPhail RC ( 1995 ) A multidisciplinary
approach to toxicological screening: III. Neurobehavioral toxicity. J
Toxicol Environ Health 45, 173-210.
Muller Popp W, G, Baltes-Schmitz B, Wehner B, Vahrenholz C,
Schmieding W, Benninghoff M, Norpoth K ( 1992 )
Concentrations of tetrachloroethene in blood and trichloroacetic acid
in urine in workers and neighbours of dry-cleaning shops. Int Arch
Occup Environ Health., 63, 393-5.
Nakai JS, Stathopulos PB, Campbell GL, Chu I, Li-Muller A, Aucoin
R ( 2000 ) Penetration of chloroform, trichloroethylene, and
tetrachloroethylene through human skin. J Toxicol Environ Health.,
58, 157-70.
Narotsky MG, Weller EA, Chinchilli VM, Kavlock RJ ( 1995 )
Nonadditive developmental toxicity in mixtures of trichloroethylene,
Di ( 2-ethylhexyl ) phthalate, and heptachlor in a 5 x 5 x 5 design.
Fundam Appl Toxicol., 27, 203-16.
Nakajima T, Kamijo Y, Usuda N, Liang Y, Fukushima Y, Kametani K,
Gonzaleza FJ, Aoyma T ( 2000 ) Sex-dependent regulation of
hepatic peroxisome proliferation in mice by trichloroethylene via
peroxisome proliferator-activated receptor alpha ( PPARalpha ).
Carcinogenesis., 21, 677-82.
Onofrj M, Thomas A, Paci C, Rotilio D ( 1998 ) Optic neuritis with
residual tunnel vision in perchloroethylene toxicity. J Toxicol Clin
Toxicol., 36, 603-7.
R.E.McCall ，F.M.A.Patel ，G.N.Mock ，P.L.Grady ；( 1998 ) Solvent on
dry washing Text.Chem.Color., 30, Nov.11
Rhomberg LR ( 2000 ) Dose-response analyses of the carcinogenic
effects of trichloroethylene in experimental animals. Environ Health
Perspect., 108, Suppl 2, 343-58.
Stacey NH ( 1989 ) Toxicity of mixtures of trichloroethylene,
tetrachloroethylene and 1,1,1-trichloroethane: similarity of in vitro to
in vivo responses. Toxicol Ind Health., 5, 441-50.
Stetkiewicz J, Zielinski K, Stetkiewicz I, Koktysz R ( 1989 ) Computer
image analysis of toxic fatty degeneration in rat liver. Pol J Occup
Med., 2, 268-78.
Sujatha TV, Hegde MJ ( 1998 ) C-mitotic effects of trichloroethylene
( TCE ) on bone marrow cells of mice. Mutat Res., 413, 151-8.
Tola S, Vilhunen R, Jarvinen E, Korkala ML ( 1995 ) A cohort study
on workers exposed to trichloroethylene. J Occup Med., Nov 22,
737-40.
Warren DA, Reigle TG, Muralidhara S, Dallas CE ( 1996 ) Schedule-controlled
operant behavior of rats following oral administration of
perchloroethylene: time course and relationship to blood and brain
solvent levels. J Toxicol Environ Health., Mar, 47: 345-62.
Waters EM, Gerstner HB, Huff JE ( 1997 ) Trichloroethylene. I. An
overview. J Toxicol Environ Health., 2, 671-707.
Wernisch M, Paya K, Palasser A ( 1991 ) Cardiovascular arrest after
inhalation of leather glue Wien Med Wochenschr., 141, 71-4.
Yoshida M, Fukabori S, Hara K, Yuasa H, Nakaaki K, Yamamura Y,
Yoshida K ( 1996 ) Concentrations of trichloroethylene and its
metabolites in blood and urine after acute poisoning by ingestion.
Hum Exp Toxicol.,15, 254-8