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.

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

Thimerosal의 발생독성에 관한 연구

곽승준(Seung Jun Kwack),이규식(Gyu Seek Rhee),김순선(Soon Sun Kim),손경희(Kyung Hee Sohn),김소희(So Hee Kim),채수영(Soo Young Chae),최요우(Yo Woo Choi),원용혁(Yong Hyuck Won),박귀례(Kui Lea Park) 한국독성학회 2003 Toxicological Research Vol.19 No.4

The purpose of our study was to evaluate the toxicity of the thimerosal in embryos and neonates. Thimerosal (also known as mercurothiolate) is a mercury-containing compound used in<br/> trace amounts to prevent bacteria and other organisms from contaminating vaccines, especially in opened multi-dose vials. The toxicity of mercury is well known and those most at risk occurrs in<br/> unborn babies and newborn babies. Test methods included in vitro whole embryo culture (WEC) system and in vivo test of neonatal toxicity in Wistar rats. Ethylmercury and methylmercury were used as positive controls for the evaluating of toxic effects of mercury. In WEC assay, treated concentrations of thimerosal, ethylmercury and methylmercury were up to 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 mg/ml, respectively. All compounds didn’t show any morphological abnormalities, but showed retardation<br/> of growth and development in dose dependent manner (>0.5㎍/ml). These data indicated that thimerosal showed developmental toxicity in vitro. In vivo neonatal toxicity, Wistar rats were administered<br/> subcutaneously with thimerosal, ethylmercury, or methylmercury (5, 25, 50, 250, and 500 mg/kg) during from postnatal day (PND) 4 to 25. Significant effects of these compounds on relative organ<br/> weights and organ morphology were not observed in this experiment. However, accumulation of mercury was detected in the kidney and testis when treated with thimerosal, ethylmercury, or methylmercury. These results suggest that thimerosal may be a harmful compound to embryo and neonate, but used concentration of thimerosal in these experiments is much higher than that of clinical application. Further investigation is needed on the safety of vaccine components, i.e. a thimerosal using in vitro and in vivo tests in the future.

LC-MS/MS를 이용한 수산물 중 니트로빈의 정량분석법 개발 및 검증

김주혜,신다솜,강희승,정지윤,이규식,Kim, Joohye,Shin, Dasom,Kang, Hui-Seung,Jeong, Jiyoon,Rhee, Gyu-Seek 한국식품위생안전성학회 2018 한국식품위생안전성학회지 Vol.33 No.2

본 연구에서는 우리나라 식품공전에서 불검출 물질로 관리하고 있는 니트로빈(nitrovin)에 대해 고감도 정량 정성분석이 가능한 LC-MS/MS를 적용하여 적합한 분석법을 제시하고자 하였다. 수산물 시료는 아세토니트릴/물로 추출하고 아세토니트릴 포화 헥산으로 지방을 제거하여 고상추출 카트리지를 적용하여 정제하였다. 분석물질은 전기분무이온화방법의 positive mode에서 이온화하여 MRM 조건을 확립하여 분석하였다. 개선된 시험법은 CODEX CAC/GL-71 가이드라인에 따라서 정확성, 정밀성, 직선성, 정량한계에 대한 검증을 통하여 유효성을 확인하였다. 본 실험에서의 정량한계는 0.001 mg/kg 수준이며, 정량한계를 포함하는 표준시료에서 얻어진 검량선의 상관계수($r^2$)는 0.985 이상으로 시험법의 직선성이 유효함을 판단할 수 있었다. 또한, 수산물(넙치, 장어 및 새우) 시료에 대한 니트로빈의 평균 회수율과 변동 계수는 72.1~122%, 2.9~16.9%로 확인되어 정확성 및 정밀성이 CODEX가이드라인에 부합하였다. 따라서, 개선된 니트로빈 정량분석법은 수산물 중 니트로빈을 분석하는데 적합하며, 니트로빈에 대한 지속적인 잔류실태조사에 활용되어 수산물 중 니트로빈의 안전관리에 기여할 것으로 판단된다. The objective of this study was to develop a sensitive method for the identification and determination of nitrovin in fishery products by using a solid-phase extraction (SPE), as performed with a liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted with a mixture of acetonitrile and water, and were then defatted with acetonitrile saturated hexane, after which further clean-up was accomplished with SPE on the hydrophilic-lipophilic balance (HLB) cartridges. The analytes were subsequently ionized in the positive mode of an electrospray ionization (ESI), and where thereby detected in a process of multiple reaction monitoring (MRM). The linearity (expressed as correlation coefficients) of the matrix calibration curves was > 0.985. The limit of the quantification for the nitrovin was measured at 0.001 mg/kg. The accuracy (expressed as average recovery) was noted between 72.1 and 122%. The precision (expressed as coefficient variation) was noted from 2.9 to 16.9%. According to the CODEX CAC/GL-71 guideline accuracy, precision, linearity, and limit of detection were determined in three matrices (which were flatfish, eel and shrimp). The proposed method was suitable for analyzing the associated nitrovin residues. This application and result can also be a factor to contribute to the non-detection drugs management in fishery products.

LC-MS/MS 및 GC-MS/MS를 활용한 수산물 중 디아제팜의 정량분석법 개발

신다솜,강희승,김주혜,정지윤,이규식,Shin, Dasom,Kang, Hui-Seung,Kim, Joohye,Jeong, Jiyoon,Rhee, Gyu-Seek 한국식품위생안전성학회 2018 한국식품위생안전성학회지 Vol.33 No.2

본 연구는 국내 생산 및 수입 양식 수산물에 대해 잔류할 수 있는 향정신성 의약품인 디아제팜 대한 안전관리강화기반을 위해 마련되었다. 중국인민공화국 국가 표준시험법(GB 29697-2013)을 기반으로 전처리 방법을 개선하여 GC-MS/MS 시험법을 확립하였으며, LC-MS/MS 방법과의 기기간 검증을 통해 확립된 시험법의 선택성, 정량한계 및 회수율에 대한 검증을 통해 디아제팜 시험법으로서의 유효성을 확인하였다. LC-MS/MS의 경우 아세토니트릴로 추출 후 PSA를 이용해 정제하였고, GC-MS/MS의 경우 아세토니트릴로 추출후 $C_{18}$카트리지를 이용해 정제하였다. 디아제팜은 표준용액을 정량한계를 포함한 농도에 따라 검량선을 작성한 결과 두 기기 모두 $r^2$> 0.99 이상의 직선성을 확인하였다. 본 실험에서의 검출한계와 정량한계는 LC-MS/MS 및 GC-MS/MS 모두 0.0004 mg/kg, 0.001 mg/kg 수준이었으며, 평균 회수율은 각각 99.8~106%, 109~124%이었다. 또한, 분석오차는 모두 15% 이하로 정확성 및 재현성이 우수하였으며, CODEX 가이드라인 규정에 만족하는 수준이었다. 따라서 개발된 시험법은 안전한 수산물의 국내 유통과 잔류실태조사를 위해 활용될 것으로 기대한다. The aim of this study was to develop an analytical method for the quantification of diazepam residues in fishery products, using liquid and gas chromatography-tandem mass spectrometry (LC-MS/MS and GC-MS/MS). The sample utilized in the study was extracted from the fish sample (crucian carp) using 0.1% formic acid in acetonitrile. For the utilization of the purification process, the dispersive solid phase extraction (dSPE) was used for LC-MS/MS, dSPE and SPE was used for GC-MS/MS, respectively. To be sure, the standard calibration curves showed a good linearity as the noted correlation coefficients, $r^2$ was > 0.99. The average recoveries for accuracy ranged in 99.8~124% for the samples which were fortified at three different levels (0.001, 0.002 and 0.010 mg/kg). The correlation coefficient for the precision effect was measured at a range of 4.01~11.8%. The limit of detection (LOD) for the diazepam analysis was 0.0004 mg/kg, and the limit of the quantification (LOQ) was 0.001 mg/kg. The proposed analytical method was characterized with a high accuracy and acceptable sensitivity to meet the established Codex Alimentarius Commission (CAC/GL71-2009) guideline requirements. We therefore established the optimal analysis method for the determination of diazepam in the fishery products using LC-MS/MS and GC-MS/MS. It would be applicable to analyze the diazepam residues in fishery products in further studies on this subject.

온라인 고체상추출과 LC-MS/MS 기술을 이용한 소변 중 파라벤류 분석

김정환,고영림,김판기,정지연,이은희,이승열,남혜선,이규식,Kim, Jung Hoan,Kho, Young Lim,Kim, Pan Gyi,Jeong, Jee Yeon,Lee, Eun Hee,Lee, Seung-Youl,Nam, Hye-Seon,Rhee, Gyu-Seek 한국환경보건학회 2012 한국환경보건학회지 Vol.38 No.6

Objectives: Parabens are widely used as antimicrobial agents in pharmaceuticals and cosmetics as well as by the food industry. Parabens have been reported to show weak estrogenic activity and be related to health effects such as allergic reactions and skin and breast cancer. We evaluated an online solid phase extraction (SPE) method coupled with LC-MS/MS technique using free and conjugated parent parabens in human urine for assessing human exposure to parabens. Methods: We employed LC/MS/MS through online solid phase extraction and column-switching techniques and analyzed free and conjugated parabens as biomarkers of human exposure. Four major parabens, methyl-paraben (MP), ethyl-paraben (EP), propyl-paraben (PP) and butyl-paraben (BP), were analyzed. Method validation was performed by sensitivity, accuracy, precision and comparison of the results of online SPE with offline SPE. Results: The limits of detection (LOD) were in the range of 0.2-2 ng/mL, and actual limits of quantification (LOQ) were in the range of 0.7-6 ng/mL urine, depending upon the compound. Accuracy was in the range of 98.3-106.4%, and precision was in the range of 1.3-8.7% (CV) depending upon the compound. We found a good correlation between the results of analysis by online SPE method and that by off-line SPE method. Conclusions: The online SPE method showed proper LOD and validated accuracy, precision and good correlation with the offline method for analyzing parabens in urine.

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.

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.

대사산물을 포함한 글리포세이트, 글루포시네이트의 분석법개선 및 잔류실태 조사

경예나 ( Yena Kyung ),이현숙 ( Hyun-sook Lee ),장문익 ( Moon-ik Chang ),이규식 ( Gyu-seek Rhee ),이한진 ( Han-jin Lee ),최은혜 ( Eun-hye Choi ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-

현재 국제식품규격위원회(CODEX)에서 글리포세이트 잔류물의 정의는 GMO인 대두, 옥수수, 유채의 경우 글리포세이트와 엔-아세틸 글리포세이트의 합으로 보고 있으며, 일반 농산물의 경우 글리포세이트로 정의하고 있으며, Estimate Daily Intake 측정시엔 글리포세이트, AMPA, 엔-아세틸글리포세이트, 엔-아세틸 AMPA를 모두 포함하도록 한다. 글루포시네이트 잔류물의 정의는 글루포 시네이트와 3-methylphosphinico propionic acid를 포함하고 있으며, 글루포시네이트에 내성을 가진 GMO 작물에 사용하는 경우 엔-아세틸 글루포시네이트를 추가하여 잔류물을 정의하고 있다. 이에 따라 본 연구에서는 글리포세이트, 글루포시네이트 모화합물과 글루포시네이트 대사산물 2종을 분석하던 현행 식품공전에서 글리포세이트 대사산물 3종을 포함한 글리포세이트 및 글루포시네이트 총 7종의 잔류실태를 조사하기 위하여 식품공전 시험법을 개선하고, 시험법 검증 및 기기 분석조건을 확립하였다. 또한 전처리시 시간이 오래 걸리는 유도체화 과정 및 정제, 농축과정을 생략· 개선하여 전처리 시간을 단축하였다. 이 화합물들은 물에 대한 용해도가 높아 물로 추출하였으며, 유지 및 비극성 갑섭 물질의 제거를 위하여 Chloroform/Dichloromethan(95/5, v/v)으로 액액분배후 원심분리하여 상층액을 취하고, Dichloromethan으로 액액분배 후 원심분리하여 상층액을 취하여 LC-MS/MS로 분석하였다. 시험법 검증을 위하여 6품목의 대표 농산물(대두, 옥수수, 감자, 현미, 감귤, 고추)을 대상으로 0.05, 0.1, 0.5 mg/kg 농도에서 회수율 실험을 한 결과 모든 처리농도 및 대상 농산물에서 70-120%의 양호한 회수율을 보였으며, 회수율에 대한 상대표준편차는 10% 이내였다. 개선된 시험법을 적용하여 국내 유통 농산물 중 옥수수와 대두를 포함한 30품목, 630건에 대하여 글리포세이트와 글리포세이트 대사산물 3종 및 글루포시네이트와 글루포시네이트 대사산물2종을 동시 분석한 결과, 글루포시네이트의 대사산물인 3-methylphosphinico propionic acid가 감자에서 2건, 양송이에서 1건, 대두에서 1건이 정량한계 이하로 검출되었으며, 나머지 626건에서는 7종 모두 검출되지 않았다.

LC-MS/MS를 이용한 농산물 중 살균제 Triforine의 시험법 검증

이현숙 ( Hyun-sook Lee ),이한진 ( Han-jin Lee ),최은혜 ( Eun-hye Choi ),장문익 ( Moon-ik Chang ),이규식 ( Gyu-seek Rhee ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-

본 연구는 최근에 단성분 검사 이력이 없으며 비교적 ADI가 낮은 살균제 Triforine을 선정하여 수입 농산물 중 잔류농약 단성분 모니터링을 하기 위하여 시험법 검증을 실시하였다. Triforine의 잔류허용기준은 곡류, 과실류, 채소류에 0.01~5.0 mg/kg으로 설정되어있다. 현재 식품공전에 기준이 설정되어 있는 농산물 외에도 추가 적용이 가능한지 미국 등 제외국 수입 의존도가 높은 상위30품목 농산물인 대두, 감자 등 콩류, 서류를 포함한 농산물에 범용적 적용 여부를 위한 시험법 검증을 실시하였다. 5종의 대표 농산물(감귤, 감자, 고추, 대두, 현미)에 0.005, 0.01, 0.05 mg/kg 농도를 적용하여 시험법 검증을 수행하였다. 회수율, 정량한계 및 분석오차는 국제식품규격위원회 (CODEX) 가이드라인에 준하여 실시하였다. 그 결과 Triforine 분석법의 정량한계는 대표농산물 5종에서 0.005 mg/kg이었고, 직선성은 0.99이상이었으며, 대표 농산물 5종에서 회수율은 74.6~108.7%이었으며, 분석오차가 10% 미만으로 시험법의 정확성 및 정밀성을 확인하였다. 본 연구에서 검증된 시험법은 수입농산물을 대상으로 잔류물의 안전관리를 위한 잔류 실태 조사 연구에 활용될 예정이다.