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1 대한응급의학회지제 21 권제 5 호 Volume 21, Number 5, October, 2010 원 저 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 충북대학교의과대학응급의학교실 훈ㆍ이석우ㆍ박정수 The Influence of Hypo-Oxygenation or Hyper-Oxygenation on Cell Survival after Paraquat Exposure in A549 Cells Hoon Kim, M.D., Suk Woo Lee, M.D., Jung Soo Park, M.D. Purpose: Paraquat (1,1 -dimethy-4,4 -bipyridinium dichloride, PQ) is a very effective and widely used herbicide that was commercially introduced in It is reduced by an electron donor such NADPH and then it transfers the electrons to molecular oxygen. As a result, the produced reactive oxygen species (ROS) are related to its cellular toxicity. However, the influence of the intracellular oxygen levels on praquat-induced oxidative cell damage has not fully been investigated. Methods: This experiment was conducted in vitro using the human carcinogenic aveolar basal epithelial cell line (A549 cells). The cytotoxicity was assessed by using the MTT method. The optical density was measured at 540 nm using an ELISA reader. We examined the morphological changes after drug treatment using an inverted microscope and fluorescence microscopy. The 2,7 -dichlorofluorescein diacetate (DCF-DA) assay was used to measure the intracellular ROS levels. Results: Incubation of the A549 cells in a hypo-oxygenation situation protected the A549 cells from paraquat-induced cytotoxicity according to the MTT & live/dead assay. The other hand, incubating the A549 cells in a hyper-oxygenation situation aggravated the PQ-induced cytotoxicity. We first 책임저자 : 훈충청북도청주시흥덕구개신동충북대학교의과대학응급의학교실 Tel: 043) , Fax: 043) nichekh2000@chungbuk.ac.kr 접수일 : 2010년 1월 25일, 1차교정일 : 2010년 2월 9일게재승인일 : 2010년 3월 11일 이논문은 2009학년도충북대학교학술연구지원사업의연구비지원에의하여연구되었음. 645 identified that the protective effects of hypo-oxygenation on the PQ-induced cytotoxicity resulted from decreased ROS production through the DCF-DA assay. Conclusion: The results of this study showed that hyperoxygenation reduced the paraquat toxicity, but hypo-oxygenation exhibits a protective effect against paraquat-induced cell death by decreasing the ROS production in A549 cells. Key Words: Paraquat, Oxygen, Tumor cell line Department of Emergency Medicine, College of Medicine, Chungbuk National University, Cheongjoo, Korea 서 파라콰트 (1,1 -dimethy-4,4 -bipyridinium dichloride) 는 1958년최초로개발된이후전세계적으로가장흔히사용되는유기염소계비선택성제초제로서그람옥산, 속사포, 파라코등의이름으로시판되고있다. 강한독성으로소량에서도인체에흡수되면치명적이며우리나라에서는연간 500명이상의사망자가발생한다고알려져있다 1). 파라쿼트의독성작용은세포내에서 cytochrome P 450 reductase, glutathione reductase 등의효소에의한파라쿼트이온의순환적환원으로활성산소종 (reactive oxygen species) 인 superoxide radicals의과다생성으로인한세포막지방과산화 (lipid peroxidation), 세포사멸 (apoptosis) 유도등에의해산화적세포손상이발생하는것으로알려져있다 2). 그러므로파라쿼트는폐, 간, 신장, 심장등신체중요장기에손상을초래하며특히폐에심한손상을일으킨다. 이는폐조직에서는에너지의존과정으로파라쿼트가흡수되어혈장과비교해폐에서의파라쿼트농도가 6~10배정도높고, 폐는대기의산소와가장가깝게접할수있는장기로서활성산소종에의해가장많이손상받기때문이다 3). 그러므로, 파라쿼트에의한활성산소종발생이폐손상의직접적원인이기때문에임상적으로파라쿼트중독시에는고농도의산소공급을하지않는것이일반적으로받아들여지고있다. 론

2 646 / 대한응급의학회지 : 제 21 권제 5 호 2010 이와반대로, 저농도의산소공급은파라쿼트중독에있어서폐손상을줄일것이라는가정을할수있다. Cheng 등 4) 의연구는 in vitro 상에서저농도의산소를전처치하는경우파라쿼트에유발된인간각막표피세포의손상을줄이는것으로보고하고있다. 또한 in vivo 연구에서도저농도산소공급이쥐의생존률을높인다고주장하였다 5). 하지만현재까지의연구들에서파라쿼트노출후가장독성이심한폐세포에서정상산소농도처리와비교하여저농도산소와고농도산소의상대적독성비교에대한 in vitro 연구가부족하며파라쿼트노출후에저농도의산소처리가세포생존과활성산소감소에미치는영향등에대한연구는거의이루어진바없다. 이에우리는인간폐암세포주에서파라쿼트로유도된세포손상에있어서고농도산소또는저농도산소처리가세포독성에미치는영향및활성산소종과의관련성을분석하기위해본실험을시행하게되었다. 대상과방법본연구의진행은인간폐암세포주에서파라콰트의농도에따른독성효과를 MTT assay 등으로분석하여독성농도를결정한후산소농도처치에따른파라콰트의인간폐암세포주에대한독성효과를알아보기위해저농도, 정상농도 ( 대기산소 ), 고농도산소를처리후에 MTT assay, live and dead cell viability assay 및현미경세포관찰등다양한분석방법으로세포독성을비교하였다. 추가적으로저농도의파라쿼트에대한세포보호효과가자유산소기감소와관련이있는지를 DCF-DA assay로분석하는실험을진행하였다. 1. 실험재료본실험에사용된파라쿼트, bovine serum albumin, dimethyl sulfoxide, tetrazolium bromide는 Sigma- Aldrich Corp (st. Louis, MO, USA) 사에서, Dulbecco s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin 등은 Gibco-BRL (Grand Island, NY, USA) 에서구입하였고, 2,7 - dichlorofluorescein diacetate (DCF-DA), live and dead cell viability assay kit는 Molecular Probes (Eugene, USA) 사제품을사용하였다. 2. 세포배양및저농도 고농도산소처리본실험에는인간폐암세포주기원인 A549 세포를사용하였으며, penicillin-streptomycin (100 ug/ml), 10% heat-inactivated FBS를첨가한 DMEM을배양액으로 5% CO2, 37 배양기에서배양하였으며, 배양접시바닥면의 70~80% 에도달하면계대배양을시행하였으며모든실험에는계대배양 10번이내의세포를사용하였다. 저농도산소처리는 99% N2/5% CO 2/1% O 2 저산소배양기 (hypoxic chamber) 에넣어 24 시간배양하였으며, 고농도산소처리는 45% N 2/5% CO 2/60% O 2 저산소배양기에 24시간넣어세포를배양하는방법으로시행하였다. 대조군 ( 대기산소 ) 은 85% N 2/5% CO 2/20% O 2 배양기에서동일조건으로배양하였다. 3. Cell proliferation assay (MTT assay) 먼저폐암세포주에서파라콰트의농도에따른독성효과평가를위해 MTT assay를시행하였다. 즉, 96-well microtitier tissue culture plate에 A549 세포를 10 3 cells/well로분주하고 10 um 파라쿼트에서 50 mm 파라쿼트농도를 8시간노출후에 PBS로 2회세척후 24시간, 48시간동안 5% CO 2, 37 배양기에서배양하였다. 이후배지를제거한후 0.5 mg/ml MTT용액 1 ml을넣고 5% CO 2, 37 C 배양기에서 2시간동안배양하였다. PBS로 2회세척한후 DMSO 200 ul를넣고 30분후 ELISA Microreader (Molecular Probes Eugene, USA) 장비를이용하여 450 nm에서흡광도를측정하였다. 다음으로, 산소농도에따른파라콰트의폐암세포주에대한독성에미치는영향을알아보기위해위에서확인된 LD 50 에가까운파라쿼트독성농도들 (0.5 mm, 1 mm) 을폐암세포주에 8시간동안노출후에 PBS로 2회세척후에고농도산소, 저농도산소, 정상산소 ( 대기산소, 대조군 ) 처리후상기 MTT assay 방식으로동일하게실시한후에흡광도를측정하였다. 상대적흡광도 (relative absorbance) 는고농도또는저농도산소에서의흡광도를정상산소 ( 대조군 ) 의흡광도로나눈값으로계산하였다. 4. Live and dead cell viability assay 및세포형태변화관찰 MTT assay에서확인된저농도에서의파라쿼트독성에대한세포보호효과를다른분석법으로확인하기위하여생존세포와죽은세포의비율을분석하기위해 live and dead cell viability assay kit (Calcein & EthD-1 염색 : 생존세포는 calcein염색으로녹색형광을, 죽은세포는 EthD-1 염색으로적색형광을보이게된다 ) 를사용하여제시된방법에따라진행하였다. 즉, 8시간의파라쿼트노출후에 24시간동안저농도산소또는정상농도산소에서세포배양후에형광염색을통해세포생존정도를확인하였다. 또한세포형태변화관찰은 inverted microscopy를이용하여세포수와형태변화양상을관찰하였다.

김 @ 훈외 : 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 / 647 A B Fig. 1. Cytotoxic activity of paraquat in A549 cells. A549 cells were treated with various concentrations of paraquat for 8 hours.

3 훈외 : 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 / 647 A B Fig. 1. Cytotoxic activity of paraquat in A549 cells. A549 cells were treated with various concentrations of paraquat for 8 hours. Cytotoxicity was determined by MTT assay, and relative absorbance comparing with control group without paraquat treatment was calculated according to doses of paraquat. (A) 24 hour after 8 hour PQ treatment, (B) 48 hour after 8 hour PQ treatment 6. 통계처리 오차범위를줄이기위해동일동시조건에서세포증식능검사를 6회시행한후평균값과표준편차를분석하였고평균값에대한 t-검정으로유의성을각각확인하였다. 결 과 Fig. 2. Effects of hypo-oxygenation on paraquat-induced cytotoxicity. A549 cells were treated with 1 mm paraquat for 8 hours and were exposed to hypoxia(1% O 2) & normoxia(20% O 2) for 24 hour, cytotoxicity was determined by MTT assay (p<0.05) 5. 세포내의활성산소측정 (DCF-DA assay) 저농도산소에서의파라쿼트독성에대한세포보호효과 가세포내활성산소의변화와관련이있는지를분석하는실험을진행하였다. DCF-DA assay는 DCF-DA가세포내에서활성산소와반응하여 DCF로변환하게되는데변화된 DCF 농도를통해활성산소발생정도를평가하는원리이다. 그러므로 8시간동안파라쿼트를처리한후독성을유발한후에 cell-permeable fluorogenic probe인 DCF-DA를각세포 well에 100 μm 씩 30분간가해준후 PBS buffer로배지에남아있는 DCF-DA를 2회세척하여제거한후정상농도또는저농도산소세포배양기에서 24시간배양하였다. 그리고나서세포내의산화과정을통해생성된 DCF를 ELISA Microreader (Molecular Probes, Eugene, USA) 로형광 (exi 480/emi530) 을측정하였다. 1. 인간폐암세포주에서의파라쿼트에의한세포독성 파라쿼트농도별로 A549 세포에미치는독성정도를평가하기위해파라쿼트를농도별로 8시간처리후에 24, 48 시간에서의세포독성을측정한결과, 농도에따른세포독성을확인할수있었다. 1 mm 농도의파라쿼트를 8시간처리후에 24시간에서흡광도감소는 42.4%, 48시간에서는 56.3% 로서세포손상이확인되었다 (Fig. 1). 이에 LD 50 에가까운 500 um, 1 mm 파라쿼트농도를고농도산소, 저농도산소처리시세포손상에미치는실험에적용하였다. 2. 파라쿼트에유발된세포독성에대한저농도및고농도산소처리의영향 8시간동안파라쿼트 1 mm를처리한후저농도 (1% 산소 ) 를 24시간처리한결과정상산소 (20% 산소 ) 에서흡광도감소가 36.7% 였는반면저농도산소에서는 9.4% 흡광도감소만이보였다 (Fig. 2). 반면고농도 (60%) 산소를처리한결과정상산소 ( 대기산소 ) 와비교하여 500 um 파라쿼트, 1 mm 파라쿼트모두에서흡광도의감소를보였다 (Fig. 3).

Effects of hyper-oxygenation on paraquat-induced cytotoxicity.

4 648 / 대한응급의학회지 : 제 21 권제 5 호 Live and dead cell viability assay 및세포형태변화관찰 8 시간동안파라쿼트 500 um 과 1 mm 을처리한후저농 도산소를 24시간처리한후세포의광학현미경을통한형태학적변화를관찰한경우저농도산소에서정상적인세포수가의미있게증가되어있었으며, 이는 live and dead Fig. 3. Effects of hyper-oxygenation on paraquat-induced cytotoxicity. A549 cells were treated with 1mM paraquat for 8 hours and were exposed to hyperoxia(60% O 2) & normoxia(20% O 2) for 24 hour, cytotoxicity was determined by MTT assay A B C D E F Fig. 4. The morphological changes of A549 cells after hypoxic or normoxic exposrue. A549 cells were incubated at 37 C for 24 hour after 8 hour PQ exposure. The morphology of the cells was observed under phase contrast microscopy ( 100). (A) Control(20% oxygen), (B) 500uM PQ(20% oxygen), (C) 1mM PQ(20% oxygen), (D) Control(1% oxygen), (E) 500 um PQ(1% oxygen), (F) 1 mm PQ(1% oxygen)

김 @ 훈외 : 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 / 649 cell viability assay를통해서도재확인할수있었다 (Fig. 4, 5).

Cells were exposed to normoxia (control), 8 hour 500 um PQ+24 hour normoxia(20% oxygen) and 8 hour 500 um PQ+24 hour hypoxia(1% oxygen).

5 훈외 : 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 / 649 cell viability assay를통해서도재확인할수있었다 (Fig. 4, 5). 즉, 세포생존을의미하는 calcein 형광염색은정상 산소에서급격히감소한반면저농도산소에서는의미있는증가를보이고있고, 동시에세포손상을의미하는 ethid- A B C A B C Fig. 5. Effect of hypo-oxygenation on cell survival with PQ-induced oxidative damage. Cells were exposed to normoxia (control), 8 hour 500 um PQ+24 hour normoxia(20% oxygen) and 8 hour 500 um PQ+24 hour hypoxia(1% oxygen). Culture dishes were subsequently stained with Molecular Probes live/dead assay and viewed under a fluorescence microscope system. (A, B) and (C) Control, 8 hour 500 um PQ+24 hour normoxia, and 8 hour 500 um PQ+24 hour hypoxia cultures, respectively, with intracellular calcein fluorescence signifying viable cells. (A ), (B ) and (C ) ethidium homodimer fluorescence of the same fields as (A, B) and (C ), respectively, which demonstrate injured and nonviable cells. Note that PQ increased cell injury/death after 24 hour, which was diminished by hypoxia Fig. 6. Dichlorofluorescein diacetate (DCF-DA) Effects of hypo-oxygenation on the production of reactive oxygen species (ROS) after various doses of PQ exposure. A549 cells were treated with PQ for 8 hours and then incubated in normoxia or hypoxia for 24 hours. Hypo-oxygenation decreased ROS production as campared with normoxia

6 650 / 대한응급의학회지 : 제 21 권제 5 호 2010 ium homodimer 형광염색은정상산소에서저농도산소보다증가된소견을보이고있다. 4. 세포내의활성산소종측정 (DCF-DA assay) 8시간동안파라쿼트를농도별로처리한후저농도와정상농도산소에서 24시간배양한 DCF-DA assay를통한활성산소종발생을측정한결과, 대부분의파라쿼트농도에서저농도산소의활성산소종발생이줄어든소견을보였다 (Fig. 6). 고찰파라콰트중독은약물중독에의한사망률에서단일원인으로는최고를차지하고있으며아직까지도적절한치료방법이확립되어있지않은상태로서전세계적으로상당한문제가되고있다. 일반적으로세포는산소이용과관련된계속적인활성산소종 (reactive oxygen species) 발생의존성세포손상이발생하게된다. 파라쿼트는이러한활성산소종생성을유발하는대표적인화합물로서세포내에들어오면 NOS oxidase, NADPH oxidase 에의해파라쿼트이온을형성하고산소와반응하여 superoxide 같은활성산소종을생성하게된다. 6) 현재까지다양한세포주에서의파라쿼트의독성평가와 in vivo 상에서의독성그리고임상에서환자에독성과관련광범위한연구가이루어지고있다 6-8). 이러한파라쿼트독성은모든장기내세포에서발생할수있으나, 특히, 폐세포는산소분압이높고자유산소기를제거하는효소계가상대적으로적어서손상에가장민감하다. 파라쿼트에의한자유산소기는폐포상피세포인 type Ⅰ, type Ⅱ폐포세포에직접작용하여폐포내출혈, 폐부종, 폐포상피세포의파괴를초래하여폐포염을유발하게되고결국폐섬유증을일으킨다. 따라서이러한파라쿼트에기인한폐섬유증은간질성폐질환의원인과병리기전을연구하는모델로서이용되고있다 9). 본실험에서는폐조직기원인인간폐암세포주 (A549) 를이용하여세포독성을조사하였다. 8시간동안파라쿼트를농도별로세포에처리한결과, 농도와시간에비례하여세포독성이있음을확인할수있었으며이는도립현미경을통한세포형태변화관찰및 calcein을이용한생존세포형광염색을통해서도유사한결과를확인할수있었다. 이러한우리의결과는많은연구들에서다양한세포주에서파라쿼트농도와시간의존성세포독성과유사한소견이다 6-9). 현재까지파라쿼트이온에의해발생되는활성산소종이파라쿼트에의한세포독성의가장중요한기전으로판단되기때문에임상적으로는파라쿼트중독환자가폐포손상 에의한호흡곤란증세가발생하더라도산소공급에의하여세포내산소농도를증가시킴으로서파라쿼트에의한산화적독성을악화시킬것으로판단되어산소치료를지양하고있다. 반대로파라쿼트독성에의한세포독성시에저농도의산소처치는활성산소종의발생이감소되어세포독성이감소할것으로판단할수있다. 하지만현재까지산소농도와파라쿼트의독성과의연관성에대한연구는그다지많지않다. 이와관련된대표적인연구로서 Hoet 등 10) 은인간 type II 폐포세포에서산소농도와파라쿼트독성에대한연구에서 LDH 분석을통해서 21% 산소농도에서 50% 세포독성파라쿼트농도로 25 um 를보인반면, 60% 산소농도에서 50% 세포독성파라쿼트농도가 7 um 로감소된소견을보였고, 10% 산소농도에서는 50% 세포독성파라쿼트농도가 1,000 um 이상으로증가되었다고보고하였다. 본연구에서는 in vitro 연구를위해비치명적인저농도산소농도를정하는예비연구를통해 1% 산소를 24시간처리하는경우저농도산소에의한세포독성이발생하지않음을확인하였다. 그리고파라쿼트를처리하고 24시간동안 20%, 60%, 1% 의산소농도를처리한후 MTT 분석뿐만아니라, 형광염색등의다양한방법을통해고농도산소에서는파라쿼트의독성이증가하고저농도의산소처치시에는세포보호효과가있다는것을확인할수있었다. 하지만현재까지파라쿼트독성에대한저농도의산소처치를통한세포보호효과가활성산소농도의감소를통해발생하는지에대한연구가이루어진바없다. 이에우리는세포내활성산소농도를측정하는실험을진행하였다. 세포내에서활성산소종을측정하는경우는 8-OH-dG 의농도측정을통해 DNA 손상정도를파악하거나세포내에존재하는지질과산화정도를측정하는방법등이주로이용되는데이러한방법들은활성산소에의해나타나는 2차적인세포내변화로서활성산소의변화량을추정해야하므로정확성이나특이성등에서제한점이많았다 11). 더구나활성산소의경우는그반감기가매우짧고세포내에존재하는다양한활성산소 scavenger에의해빠른시간내에복구가일어나므로가능한빠른시간내에생성된활성산소를측정하는것이정확성을높이는중요한관건이될것이다. Keston과 Brandt 12) 는 2 7 -dichlorodihydrofluorescein diacetate (DCF-DA) 를세포배양액에첨가하게되면세포내의활성산소에의한산화과정을통해서 2 7 -dichlorofluorescine으로변화되는이것이형광을발생하므로특정파장에서의형광의정도로서활성산소의양을직접정량할수있는방법을보고하였는데본연구는이들의방법을변형하여활성산소발생정도를측정하였다. 이를통해서저농도의산소공급에서정상산소에서보다활성산소발생이감소된것을확인할수있어서저농도산

7 훈외 : 인간폐암세포주에서파라쿼트노출후에저농도또는고농도산소처리가세포생존에미치는영향 / 651 소의세포보호효과일부는활성산소감소와관련이있다는사실을처음으로확인할수있었다. 이로서본연구를통해 in vitro연구에서저농도의산소공급이파라쿼트독성을줄이는데매우효과적인방법임을확인할수있었으며, 일부는활성산소감소와관련이있다는것을확인할수있다. 향후파라쿼트독성에대한동물모델연구를통한보호효과연구및활성산소외에다른세포보호기전에대한추가적인연구를통해저농도산소를통한파라쿼트치료에대한심도있는연구가필요할것으로사료된다. 결 이번연구는인간폐암세포주에서파라쿼트의세포독성과관련되어저산소및고산소처리가세포독성에미치는효과를분석한것이다. 먼저 A549세포에서파라쿼트의세포독성은농도와시간에비례적으로증가하는양상을보였고저농도산소처치는파라쿼트에의한세포보호효과가높다는것을확인하였으며이러한효과는활성산소감소와일부관련이있다는것을증명하였다. 론 참고문헌 01. Lee EY, Kim YT, Yang JO, Hong SY. Long-term prognosis of paraquat-induced lung injury. Korean J Med 2003;65: Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology 2002;180: Lee SI, An KW, Chung CH. Effects of aminotriazole on lung toxicity of paraquat intoxicated mice. Tuberc Respir Dis 1994;41: Cheng Q, Nguyen T, Song H, Bonanno J. Hypoxia protects human corneal endothelium from tertiary butyl hydroperoxide and paraquat-induced cell death in vitro. Exp Biol Med (Maywood) 2007;232: Rhodes ML, Zavala DC, Brown D. Hypoxic protection in paraquat poisoning. Lab Invest 1976;35: Bonneh-Barkay D, Reaney SH, Langston WJ, Di Monte DA. Redox cycling of the herbicide paraquat in microglial cultures. Brain Res Mol Brain Res 2005;134: Tuschl T, Schwab CE. The use of flow cytometric methods in acute and long-term in vitro testing. Toxicol In vitro 2005;19: Choi YI, Noh EW, Han MS, Yi YS. Estimation of cellular damage caused by paraquat and lead using a cell culture system. J Plant biotechnology 2001;3: Park JK, Kim GT, Song HS, Park SM. Apoptosis in respiratory epithelial cells: triggering by paraquat and modulation by L-ascorbic acid. J Korean Soc Emerg Med 2004; 15: Hoet PH, Demedts M, Nemery B. Effects of oxygen pressure and medium volume on the toxicity of paraquat in rat and human type II pneumocytes. Hum Exp Toxicol 1997;16: Huh EH, Lee JK, Han SH. The effects of Ginkgo biloba extract (EGb 761) on ethanol-induced cytotoxicity in PC12 cells. Dementia and Neurocognitive Disorders 2004;3: Keston AS, Brandt R. The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Anal Biochem 1965;11:1-5.

06-12김 훈

06-12김 훈 대한응급의학회지제 18 권제 1 호 Volume 18, Number 1, February, 2007 원 저 항산화제 Quercetin 이파라쿼트에유도되는세포손상에미치는효과 충북대학교의과대학응급의학교실 김 @ 훈ㆍ이석우 The Effects of Quercetin on Paraquatinduced cell Damage Hoon Kim, M.D., Suk-Woo