p53-아데노바이러스 벡터 복강투여에 따른 마우스에서의 생식독성 평가

이규식(Gyu-Seek Rhee),이현주(Hyun-Joo Lee),손경희(Kyung-Hee Sohn),김순선(Soon-Sun Kim),곽승준(Seung-Jun Kwack),이이다(Rhee-Da Lee),원용혁(Young-Hyuk Won),조대현(Dae-Hyun Cho),이승훈(Seung-Hoon Lee),박귀례(Kui-Lea Park) 대한미생물학회 2004 Journal of Bacteriology and Virology Vol.34 No.1

아데노바이러스 유전자치료벡터의 생식독성 연구

이규식,곽승준,김순선,이이다,석지현,채수영,정수연,김승희,이승훈,박귀례,Rhee, Gyu-Seek,Kwack, Seung-Jun,Kim, Soon-Sun,Lee, Rhee-Da,Seok, Ji-Hyun,Chae, Soo-Young,Chung, Soo-Youn,Kim, Seung-Hee,Lee, Seung-Hoon,Park, Kui-Lea 한국미생물학회 2007 미생물학회지 Vol.43 No.3

유전자치료W터의 주입시 생식세포를 통한 다음 세대로의 전달 가능성은 안전성 측면에서 관심을 중대시키고 있다. 특히 전립선암이나 난소암의 치료시 바이러스를 생식기관에 인접한 부위에 주입하여야 하므로 그 가능성이 높다. 따라서 본 연구에서는 유전자치료에 많이 이용되는 아데노바이러스를 매개로하여 tumor suppressor 유전자인 p53을 발현하는 아데노바이러스 벡터를 제조하여 이를 투여시 생식장기를 포함한 주요장기조직에의 분포와 germ cell을 통한 차세대로의 전달 가능성 등의 생식독성을 조사하였다. In vivo biodistribution study를 위하여 $Ad-CMV-{\beta}-gal$흑은 Ad-CMV-p53를 마우스 암 수의 복강에 주사한 후 생식장기를 포함한 주요 장기에서 아데노바이러스 유래 DNA검출 및 RNA발현 여부를PCR과 RT-PCR로 각각 확인하였다. 그 결과 간 및 비장과 같은 일반 장기에서도 주입한 외부유전자의 DNA가 검출되거나RNA가 발현되었을 뿐만 아니라, 정낭, 전립선, 부고환, 난소 및 자궁 등의 생식장기에서도 주입한 외부유전자가 검출되거나 발현되는 것으로 나타났다. Real-time PCR을 이용하여 각 장기에서의 투여된 아데노바이러스 벡터는 시간 의존적으로 감소되는 것을 정량하였다. Ad-CMV-p53를 암 수 마우스의 난소와 고환에 각각 직접 주사하여 교배시킨 후 그 후세대의 DNA를 분리하여 주입한 아데노바이러스 유래의 DNA를 검색한 결과, 어떠한 차세대에서도 주입한 아데노바이러스 유래의 DNA가 검출되지 않았다. 한편 생식장기에서의 PCR및 RT-PCR signal유래 vector의 위치를 확인하기 위해 매우 감도가 높은 in-situ PCR로 조사한 결과 고환의 경우 간질조직으로의 전달은 일어나나 정세관 내에는 아데노바이러스 벡터가 전달되지 않으며, 난소에서도 아데노바이러스벡터는 난포내의 난자에 전달되지 않고 기질조직에 존재하는 것으로 확인되었다. 결론적으로 복제 능력 이 결여된 아데노바이러스를 매개로 한 유전자치료제는 생식 장기에서 검출되더라도 다음 세대로 전달될 가능성은 대단히 낮음을 제시한다. The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

위염치료제의 임상시험평가지침 연구

송윤경,진선경,한의식,안미령,정주연,이이다,조일영,김동섭,지은희,박효영,오정미,신원,이선희,김인규,Song, Yun-Kyoung,Jin, Sun-Kyung,Han, Eui-Sik,Ahn, Mee-Ryung,Jung, Ju-Yeon,Lee, Rhee-Da,Cho, Il-Yong,Kim, Dong-Sub,Ji, Eun-Hee,Park, Hyo-Young,Oh, 대한약학회 2011 약학회지 Vol.55 No.4

Gastritis is the most common disease among Korean. The demand for the development of gastritis drugs has been increasing. Currently, however, there is no guideline available for the clinical evaluation of gastritis drugs worldwide. As a consequence, domestic and international pharmaceutical companies make errors in the drug development processes, and it becomes difficult for them to establish the scientific validity and objectivity of newly developed drugs. The objective of this study was to develop the Guideline for Clinical Trials Evaluation of Gastritis which can be used in improving the quality and consistency of clinical trials. First, we collected and reviewed the clinical trials on gastritis drugs that were available from Japan Pharmaceuticals and Medical Devices Agency and Korea Food and Drug Administration (KFDA), and investigated the recent research trends on clinical trials of gastritis drugs. Reviewers from KFDA and National Institute of Food and Drug Safety Evaluation and scientific experts from the pharmaceutical industries developed the guidelines through regularly scheduled meetings. Opinions and consultation from academic fields and industry experts were also obtained. This project will provide the clinical trial practitioners, investigator and reviewers the scientific and rational guidelines for performance and evaluation of clinical trials for gastritis drugs. Furthermore, we hope this guideline contributes to establishing the national competitiveness, improving the quality of clinical trial, and encouraging researches on drug development for gastritis.

E-screen assay 및 자궁비대반응시험(Uterotrophic assay)을 이용한 di-(2-ethyIhexyl) adipate의 에스트로겐성 작용에 관한 연구

한순영(Soon Young Han),김형식(Hyung Sik Kim),한상국(Sang Kook Han),이이다(Rhee Da Lee),양규환(Kyu Whan Yang),박귀례(Kui Lea Park) 한국식품과학회 2000 한국식품과학회지 Vol.32 No.4

3-MCPD의 생식˙발생독성에 관한 연구

곽승준(Seung Jun Kwack),김순선(Soon Sun Kim),최요우(Yo Woo Choi),이규식(Gyu Seek Rhee),손경희(Kyung Hee Sohn),이이다(Rhee Da Lee),채수영(Soo Young Chae),정용현(Yong-Hyun Chung1),유일재(Il Je Yu1),박귀례(Kui Lea Park) 한국독성학회 2004 Toxicological Research Vol.20 No.2

3-Monochloro-1,2-propanediol(3-MCPD) is a toxic compound, often present in different foods containing acid hydrolyzed(AH) protein, like seasonings and savory food products. The purpose of the present study was to investigate the effects of 3-MCPD on male fertility, sperm and testosterone secretion. In vivo male fertility test was performed for observing the adverse effects of 3-MCPD on the function of male reproductive system and pregnancy outcome. 0.01, 0.05, 0.25, 1 and 5 mg/kg b.w. of 3-MCPD was given daily by gavage to groups of 15 adult male SD rats for 4 weeks. At the end of pre-treatment period, males were mated overnight with normal females. Following morning, males demonstrating successful induction of pregnancy were sacrificed on that day to assess sperm parameters and histopathology of reproductive organs. The resulting pregnant females were sacrificed on day 20 of gestation to evaluate pregnancy outcome. As a result, four-week paternal administration with 3-MCPD resulted in adverse effects on male fertility and pregnancy outcome without remarkable histopathological changes in testes and epididymides; sperm motility, copulation index and fertility index were markedly decreased in the treated group and numbers of live fetuses showed steep dose-response curves. Also, spermatogenesis was investigated in this experiment. However, no<br/> effect was observed on production of sperm in testes treated with 3-MCPD for 4 weeks. Hormone assay was performed for observing the effects of 3-MCPD on testosterone and luteinizing hormone (LH) in blood and testes of male SD rats and cultured primary Leydig cell. In result, significant changes of related hormones did not observed by treatment of 3-MCPD. These results indicated that paternal treatment with 3-MCPD induced spermatotoxic effect, which caused an antifertility on male.

3-MCPD의 생식ㆍ발생독성에 관한 연구

곽승준(Seung Jun Kwack),김순선(Soon Sun Kim),최요우(Yo Woo Choi),이규식(Gyu Seek Rhee),손경희(Kyung Hee Sohn),이이다(Rhee Da Lee),채수영(Soo Young Chae),정용현(Yong-Hyun Chung),유일재(Il Je Yu),박귀례(Kui Lea Park) 한국독성학회 2004 Toxicological Research Vol.20 No.1

3-Monochloro-1,2-propanediol(3-MCPD) is a toxic compound, often present in different foods containing acid hydrolyzed(AH) protein, like seasonings and savory food products. The purpose of the present study was to investigate the effects of 3-MCPD on male fertility, sperm and testosterone secretion. In vivo male fertility test was performed for observing the adverse effects of 3-MCPD on the function of male reproductive system and pregnancy outcome. 0.01, 0.05, 0.25, 1 and 5 mg/kg b.w. of 3-MCPD was given daily by gavage to groups of 15 adult male SD rats for 4 weeks. At the end of pre-treatment period, males were mated overnight with normal females. Following morning, males demonstrating successful induction of pregnancy were sacrificed on that day to assess<br/> sperm parameters and histopathology of reproductive organs. The resulting pregnant females were sacrificed on day 20 of gestation to evaluate pregnancy outcome. As a result, four-week paternal<br/> administration with 3-MCPD resulted in adverse effects on male fertility and pregnancy outcome without remarkable histopathological changes in testes and epididymides; sperm motility, copulation index and fertility index were markedly decreased in the treated group and numbers of live fetuses showed<br/> steep dose-response curves. Also, spermatogenesis was investigated in this experiment. However, no effect was observed on production of sperm in testes treated with 3-MCPD for 4 weeks. Hormone assay was performed for observing the effects of 3-MCPD on testosterone and luteinizing hormone (LH) in blood and testes of male SD rats and cultured primary Leydig cell. In result, significant changes of related hormones did not observed by treatment of 3-MCPD. These results indicated that paternal treatment with 3-MCPD induced spermatotoxic effect, which caused an antifertility on male.

흰쥐 배양 전배자 및 중뇌세포에서 Ochratoxin A의 독성

홍진태(Jin Tae Hong),박귀례(Kui Lea Park),한순영(Soon Young Han),박기숙(Ki Sook Park),김형식(Hyung Sik Kim),오세동(Se Dong Oh),박희정(Hee Jung Park),이이다(Rhee Da Lee),장성재(Seung Jae Jang) 대한약학회 1998 약학회지 Vol.42 No.3

Effects of ochratoxin A (OTA) on embryo development were studied in cultured whole embryos from 9.5 day gestation rat for 48 h. OTA (more than 0.5mcg/ml) induced microcephaly in the cultured rat whole embryos. Protein and DNA content, and DNA synthesis were significantly inhibited by OTA. We next examined whether the microcephaly seen in cultured whole embryo partially results from inhibition of differentiation of embryonic midbrain cells. Embryonic midbrain cells were extracted from 12 day gestation rat embryos, and cultured for 96 hr. OTA ibhibited cell differentiation about 50% over control. We also tested whether OTA-induced embryotoxicity would be associated with oxidative damages. We measured the gamma-glutamyltranspeptidase (gamma-GT) and glutathione peroxidase (GPX) activities, and glutathione (GSH) content in both cultured whole embryos and embryonic midbrain cells. OTA decreased GSH content, whereas slightly increased gamma-GT activity, but GPX activity was not significantly changed. These results show that OTA caused the microcephaly and its effect may be partially due to the inhibition of cell differentiation of embryonic midbrain cells, but the role of oxidative damages is not clear in embryotoxicity.

Natural Toxin의 안전성 평가연구 : Ochratoxin A의 생식독성에 관한 연구 Reproductive toxicity of ochratoxin A

홍진태,박귀례,한순영,박기숙,김형식,오세동,박희정,최윤진,국선숙,이이다,장성재 식품의약품안전청 1997 식품의약품안전청 연보 Vol.1 No.-

Ochratoxin A (OTA)는 Aspergillus와 PeBicillum속에 속하는 진균룬가 생성하는 mycotoxin의 일종으로 엇드, 마우스 및 햄스터를 이용한 invivo시험에서 태자의 체중감소, 골화지연 및 중추신경계 이상이 보고되었다. 본 실험에서는 전배자배양법을 이용하여 OTA가 embryo 발달에 미치는 영향에 대하여 연구하였으며 embrl·onic mid-brain cells를 이용하여 embryot()xicity의 작용 메카니즘을 연구하였다. 임신 9.5일째 rat whole embryo에 07,'1(0.5-4tg/mf)를 48 시간 노출시킨 결과 쇼두증(microcephalys)을 일으켰으며, 소두증을 일으키는 농도 (1 및 3f g/mf)에서 whole embryo의 protein 및 DNA함략이 감소하였고 DNA합성이 저하되었다. 소두증이 embryonic midbrain cells의 분화 및 증식억제에 의한 결과인지를 알아보기 위하여 임신 12일째 rat embrl·o에서 midbrain cells를 분리하여 96시간 OTA에 노출시킨 결과 0.Sfg/mf에서 까포 분화를 50% 억제하였고 세포증식도 저왜귀였다. Smbryotoxicity가 OTA에 의해 생성되는 활성산소에 의한 산회적 손상 때문인지를 알아보기 위하여 whole embryo (1.5 및 3rg/mf OTA:i와 midbrain cells (0.25와 0.Srg/mf OTA)에서 OTA엔 의한 f-91u1am?t- transpeptidase activity (γ-GT), glutathione peroxidase (GP보j activ쓿y 및 91utathione content변화를 측정한 결과 γ-GT는 약간 증가한 반면 glutathione 함량은 감소하였고 GP린 활성은 변화가 없었파, 이상의 결과는 1) OTA는 0.Sr9/m1이상의 농도에콕 소두증을 일으키는 embryotoxic하며 2) m전brain 세포분화 띤 증식읔제rl소두증을 일으키는 기전에 부분적으로 작용한다고 생각되며 3) 07.4의 embryoto온c균y에 있어서 산화적 손상의 역할에 대하여는 더 연구가 필요하다핏 판단된다. Teratogenic effects of ochratoxin A (OTA) were studied in cultured rat whole embryo from day 9.5 gestation for 48 hrs. All embryos exposed to more than 0.Sfg/mj OTA weredevelopmentally defects. T:he mest effect was seen in head causiug microcephaly. Protein and DNA con-tent, and DNA syntllesis were significantly inhibited by OTA. In separated experiment. we eBaminedwhether the microcephaly seen in cultured whote embryo partially results from inhibition ofdiffereBtiation of midbrai:R cetls. Embryonic midbrain cells were extracted from day 12 gestation rat em-bryos, and cultured for 96 hrs. 0_Sfe/mf OTA inhibited cell differe·ntiation about 50% over control, andalso iBhibited cell proliferation significantly, but did Bet change DNA and protein conteBt. We also test-ed whether OTA-induced toxicity in embryos would be associated rrith oxidative damages. We measuredthe gamma- glutamyltranspeptidase ( γ-fT ) and glutathione peroxidase ( GPX ) activites, and glutathi-one (eSH ) content in both cultured whole embryos and embryonir midbraiB cells. OTjt decreased GSHcontent, but slightly increased γ-GT activity, whereas GPX activity was not significantly changed.These ?efults show that OTA caused the microcephaly and its effect may be partially due to the inhibi-tion of cell differentiatioil of embryonie midbrain cells, but the role of oxidative damage is not clear inernbryotoxicity.