담배 흡입독성 평가를 위한 설치류 담배연기노출시스템의 유효화

손형옥,이형석,신한재,박철훈,유지혜,장미,현학철,Sohn, Hyung-Ok,Lee, Hyeong-Seok,Shin, Han-Jae,Park, Chul-Hoon,Yoo, Ji-Hye,Jang, Mi,Hyun, Hak-Chul 한국연초학회 2014 한국연초학회지 Vol.36 No.1

As part of a balanced testing battery, subchronic inhalation studies on rats are performed to ensure that proposed cigarette modifications do not increase the toxicity of smoke and to demonstrate any instances where a modification may actually contribute to harm reduction. For subchronic inhalation studies with aerosols, the OECD suggests an exposure regimen of 6 hours/day (OECD Guideline 413, 1981), but alternative regimens have also been published: 1 hour/day and $2{\times}1$ hour/day. The aim of this study was to validate a rodent nose-only exposure system for the assessment of inhalation toxicity of cigarette smoke. In this study, cigarette smoke exposure system is consisted of cigarette smoke generator, smoke concentration adjusting system, and 20-port nose-only exposure system. Male SD rats were exposed for 35 days ($2{\times}1$ hour/day) to 3R4F Reference cigarette smoke and analysed major monitoring items of OECD Gudeline 413. WTPM, was measured in the test atmosphere, respiratory function (Buxco Biosystems) during exposure, postexposure urinary exposure biomarkers and alveolar neutrophiles in BAL fluid (Day 35) were evaluated. Validation demonstrated steady WTPM ($257{\pm}20ug/L$, $502{\pm}27ug/L$) and spatial uniformity (<10%). Nose port temperature ($22{\sim}26^{\circ}C$ and RH (45~75%) were acceptable over 35 days. Reductions in respiratory rate and minute volume and increase in the neutrophiles in BALF and the urinary exposure biomarkers were observed cigarette smoke dose dependently. This validation and 35-day inhalation study has shown that the rodent nose-only exposure system may be useful in the inhalation toxicity assessment of cigarette smoke.

담배연기응축물의 소핵생성 측정시 두가지 방법간의 민감성 비교

손형옥,이영구,한정호,허재연,이동욱,현학철,신한재,Sohn Hyung-Ok,Lee Young-Gu,Han Jung-Ho,Hur Jae-Yeon,Lee Dong-Wook,Hyun Hak-Chul,Shin Han-Jae 한국연초학회 2004 한국연초학회지 Vol.26 No.2

Among short-term in vitro genotoxicity assays, micronucleus assays are rapid, inexpensive, and less labor-intensive system. We have undertaken a comparative study of sensitivity of cigarette smoke condensate(CSC) by general micronucleus(MN) assay and cytokinesis-block micronucleus(CBMN) assay. In this study, V79 Chinese hamster cells were employed to evaluate and compare the genotoxicity of CSC of Kentucky Reference Cigarette 2R4F by 2 kinds of in vitro MN assay methods. To determine the optimum concentration of cytochalasin B(CYB) to obtain the maximal number of binucleated cells for CBMN assay, triplicate cultures of growing cells were treated with CYB for 15 h. CYB treatments caused a concentration-dependent increase in cytotoxicity($1\~4{\mu}g/mL$) and proportion($0.25\~1\;{\mu}g/mL$) of binucleated cells. These data suggested that 1 ug/mL of CYB is as an optimum dose for CBMN assay in binucleated V79 cells. Short treatment(4 h) of CSC induced a micronucleated cells with a concentration-dependent response in the presence or absence of CYB, but CSC-induced MNs were weakened when S9 was present. Long treatments(19 h) of CSC also induced a significant increase MN formation with a concentration-dependent response. At a concentration of 75 ${mu}g/mL$, the MN cell frequencies of general MN assay and CBMN assay were $6.5\%\;and\;11.7\%$, respectively. Linear regression analysis revealed a good correlation in CBMN assay between a concentration of CSC and MN cell frequency. All these data indicated that CBMN assay is more sensitive to the induction CSC-induced MN than general MN assay.

담배적용 생체내(in vivo) 평가;아만성 흡입독성 시험을 중심으로

손형옥,Sohn, Hyung-Ok 한국연초학회 2008 한국연초학회지 Vol.30 No.1

생물학적으로 활성이 있는 물질들의 생체 내 대사를 이해하고 인체 독성 및 질환의 발현을 예측할 수 있는 가장 좋은 시험방법은 생체 내 독성 평가법이다. 담배연기의 생체 내 독성 평가법에는 14일 흡입독성시험, 90일 흡입독성 시험 및 피부도말 종양발생 시험 등이 있다. 90일 흡입독성 시험은 담배의 일반적인 독성 정보를 제공한다. 생체 내 독성평가는 담배 첨가물, 재료품 또는 제품 설계를 변경할 경우 담배연기 독성이 증가 또는 감소되었는 지를 평가할 수 있으며 이 결과는 제품보증에 활용될 수 있다. 캐나다의 독성평가자료 제출 및 EU 등의 담배첨가물에 대한 규제는 향 후 이런 생체 내 시험에 대한 지속적이고도 구체적인 요구를 할 것으로 사료된다.

흰쥐 간 마이크로좀의 Cytochrome P - 450 과 Testosterone 및 16α

손형옥,임흥빈,이영구,이동욱,박희윤 ( Hyung Ok Sohn,Heung Bin Lim,Young Gu Lee,Dong Wook Lee,Hee Yun Park ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.4

The binding affinity of the differentially induced cytochrome P-450 (P-450) in rat liver microsomes with testosterone and 16α-hydroxytestosterone (16α-OH-T) was compared. Interaction of testosterone with all three microsomel P-450 of normal, MC- or PB-treated rats, gave type I spectral changes and their spectral binding constants (Ks) to P-450s were 35, 33, and 25 μM at 25℃, respectively. PB-inducible P-450s among three of these showed the highest affinity to testosterone, and the ratio of low to high spin conversion of P-450 by this compound showed also a similar tendency as the results of binding affinity. 16α-OH-T, one of testosterone metabolites, binding to all three P-450 exhibited a reversed type I spectral changes and their Ks values to normal, MC- and PB-inducible P-450 were 41, 28, and 23 μM, respectively, but binding of it with PB-inducible P-450 showed another Soret band near 460 nm. These results indicate that 16α-OH-T also binds to P-450s with a high affinity like testosterone, and it may be further metabolized to more polar compounds by P-450.

35종의 특정 화학성분들의 in vitro 활성 평가

신한재,손형옥,박철훈,이형석,민영근,현학철,Shin, Han-Jae,Sohn, Hyung-Ok,Park, Chul-Hoon,Lee, Hyeong-Seok,Min, Young-Keun,Hyun, Hak-Chul 한국연초학회 2007 한국연초학회지 Vol.29 No.1

The objective of this study was to investigate the contribution of various smoke constituents to the toxicological activity of total particulate matter(TPM) or the gas/vapor phase(GVP). These components included phenol compounds, aromatic amines, polycyclic aromatic hydrocarbons, heterocyclic amines, and carbonyl compounds. The mutagenic and cytotoxic potencies were assessed using the Salmonella mutagenicity assay with S. typimurium TA98 strain and the neutral red uptake cytotoxicity assay(NRU) with BALB/c 3T3 fibroblast cells, respectively. The Salmonella mutagenicity test showed that heterocyclic amines exhibited significantly higher levels of toxicity compared to other smoke constituents. Among them, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ) was shown the most mutagenic compound with a specific mutagenicity of $7.9{\times}10^5\;revertants/{\mu}g$. An analysis of the possible contribution revealed that MeIQ account for only 0.85% of the 2R4F-TPM mutagenicity in TA98. NRU data demonstrated that high cytotoxic activity was obtained for hydroquinone, formaldehyde, and acrolein. Based on the results of the present study, the contribution of acrolein to the cytotoxicity of the GVP fraction was calculated as 61%. Thus, a large proportion of the cytotoxic activity of this complex mixture, cigarette smoke gas phase, can be attributed to the acrolein.

인간 유래 폐 세포주별 담배연기 분획의 염증 반응 민감도 비교

유지혜,손형옥,박철훈,이형석,장미,현학철,신한재,Yoo, Ji-Hye,Sohn, Hyung-Ok,Park, Chul-Hoon,Lee, Hyeong-Seok,Jang, Mi,Hyun, Hak-Chul,Shin, Han-Jae 한국연초학회 2010 한국연초학회지 Vol.32 No.1

The aim of this study is to compare the sensitivity of both two NCI-H292 and A549 cell types to acute inflammatory responses induced by cigarette smoke. For this, we treated two kinds of smoke fractions derived from 2R4F reference cigarettes: total particulate matter(TPM) collected onto a Cambridge filter pad and gas/vapor phase(GVP) prepared by bubbling through in buffer solution. When we measured cellular cytotoxicity by neutral red uptake assay after treatment for 24 hours, TPM and GVP induced cytotoxic effect in a dose-dependent manner in the range of 10-$100{\mu}g$/mL and 60-$300 {\mu}g$/mL., respectively, in both cell types without any cellular difference. Additionally, when we examined acute inflammatory responses by analyzing cytokines secreted into culture media including tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interleukin-8(IL-8), and transforming growth factor-$\alpha$(TGF-$\alpha$) as well as matrix metalloproteinase-1(MMP-1), the treatment with smoke fractions increased those marker proteins in a dose-dependent manner in NCI-H292. Meanwhile, in A549 cells only MMP-1 was observed to be increased in a dose-dependent fashion. Collectively, our data indicate that NCI-H292 cell type is more sensitive to cigarette smoke-induced inflammatory response than A549 cells. This suggests that NCI-H292 could be useful as an in vitro evaluation tool to assess harmful effects of cigarette smoke.

질소 화합물이 담배연기성분 및 안전성에 미치는 영향

신한재,박철훈,손형옥,이형석,김용하,현학철,Shin, Han-Jae,Park, Chul-Hoon,Sohn, Hyung-Ok,Lee, Hyeong-Seok,Kim, Yong-Ha,Hyun, Hak-Chul 한국연초학회 2009 한국연초학회지 Vol.31 No.2

The objective of this study was to investigate the effect of nitrogen compounds such as protein on the chemical composition and toxicity of cigarette mainstream smoke. BSA protein was treated into the tobacco leaf of original 2R4F cigarette at 1~4 % level. The studies were performed which included a bacterial mutagenicity assay and a mammalian cell cytotoxicity assay for total particulate matter(TPM), and glutathione(GSH) consumption assay for gas/vapor phase(GVP) and determination of smoke chemical constitute. Cigarettes treated with protein were observed dose-dependent increase in yield of volatiles, semi-volatiles and aromatic amines compared with control cigarette. However, carbonyl compounds such as acrolein was lower than that of control cigarette when calculated on an equal TPM basis. The cytotoxicity of TPM obtained from the protein-added cigarettes was not different from that of control cigarette. However, the mutagenicity of the TPM from protein-treated cigarettes(1~4 %) was up to 10-27 % higher than that of control. On the other hand, toxicity of GVP from protein-treated cigarette(4 %) was significantly decreased compared with control cigarette. An overall assessment of our data suggests that nitrogen compounds such as protein should be important for the chemical composition and biological activity of cigarette mainstream smoke.

담배 주류연의 생물학적 활성에 대한 흡연조건의 영향

신한재,박철훈,손형옥,이형석,유지혜,이병찬,현학철,Shin, Han-Jae,Park, Chul-Hoon,Sohn, Hyung-Ok,Lee, Hyeong-Seok,Yoo, Ji-Hye,Lee, Byeong-Chan,Hyun, Hak-Chul 한국연초학회 2008 한국연초학회지 Vol.30 No.1

The objective of this study was to determine the effect of smoking conditions on the in vitro toxicological activity of mainstream smoke. The 2R4F reference cigarette was machine-smoked by International Organization for Standardization(ISO) and Canadian Intense(CI) conditions. Smoke was analysed for chemical composition and in vitro toxicity. The cytotoxic potencies of both the total particulate matter(TPM), which were collected in Cambridge filter pad, and gas/vapor phase(GVP), which was bubbled through in phosphate-buffer saline in a gas-washing bottle, were assessed neutral red up take assay with chinese hamster ovary(CHO) cells. The assessment for genotoxicity of TPMs generated under ISO and CI conditions was determined using Salmonella mutagenicity assay and in vitro micronucleus assay. When calculated on an equal TPM basis, in vitro toxicity of TPM obtained under CI condition was decreased compared to TPM generated under ISO condition. The results of chemical composition analyses revealed that the lower toxicological activity under CI condition than that of ISO condition could be explained by the decreased in the contents of phenols, N-nitrosoamines and aromatic amines of TPM on an equal TPM basis.

담배연기 노출량 평가 생체지표

박철훈,신한재,이형석,유지혜,손형옥,Park, Chul-Hoon,Shin, Han-Jae,Lee, Hyeong-Seok,Yoo, Ji-Hye,Sohn, Hyung-Ok 한국연초학회 2009 한국연초학회지 Vol.31 No.1

Biomarkers could be critical and useful tools for assessing the biological effects of smoking and detecting differences between potentially reduced exposure product (PREP) and conventional cigarettes. Smoking-related biomarkers can be classified into three categories as biomarkers of exposure, biomarkers of effects, and biomarkers of potential harm. When compared with the biomarkers of effects or harm, the biomarkers of exposure for chemical constituents of cigarette smoke are well established and characterized. In addition, they could offer the important information in understanding how cigarette smoke interacts with biological molecules and causes the disease to human. Therefore, we provide an overview of 6 biomarkers of exposure (Nicotine and nicotine metabolites, Carboxyhaemoglobin, NNAL (4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol) and NNAL - glucuronide, 3-Hydroxypropyl-mercapturic acid, and Monohydroxy-butenyl-mercapturic acids, and Urine mutagenicity) which were validated through extensive research and clinical experience. These reliable biomarkers could help identify the efficacy of PREP by predicting early toxicological effects and lead to improve it.

랫드에서 TCDD 투여에 의해 유도된 생체독성의 고려홍삼 추출물에 의한 억제 효과

최수진(Soo Jin Choi),손형옥(Hyung Ok Sohn),신한재(Han Jae Shin),현학철(Hak Cheol Hyun),이동욱(Dong Wook Lee),송용범(Yong Bum Song),이수현(Soo Hyun Lee),강동호,임학섭(Hak Seob Lim),이철원(Cheol Won Lee),문자영(Ja Young Moon) 고려인삼학회 2008 Journal of Ginseng Research Vol.32 No.4

TCDD가 실험동물에 노출되었을 때 유발되는 생체독성을 예방 또는 억제할 수 있는 고려홍삼 추출물의 효과를 탐색하였다. 이를 위하여 TCDD(25μg/kg bw, 1회 투여)와 홍삼추출물(100 mg/kg bw, 격일투여)을 각각 단독 또는 병행 복강 투여한 다음 32일 동안에 체중과 각 장기들의 무게의 변화, 뇨 분석, 혈액학적 및 혈액화학적 변화를 관찰하였다. TCDD 의 단독투여에 의하여 체중의 증가정도가 정상군 또는 홍삼 단독 투여군의 체중증가율에 비하여 상당히 감소하였다. TCDD와 홍삼추출물을 병행 투여한 흰쥐에서의 체중증가율은 TCDD 단독투여에 의해 감소된 체중증가율을 다소 회복 시키는 결과를 나타내었다. TCDD를 단독 투여한 실험군에서는 간의 무게가 TCDD 투여 후 2, 5, 및 16일째에 대조군에 비하여 특이적으로 증가하는 특징을 보였으나, 홍삼추출물을 단독 투여한 실험군에 서의 간의 무게는 대조군의 간의 무게에 비하여 다소 감소하였다. TCDD와 홍삼추출물이 병행 투여된 흰쥐에서의 간의 무게의 증가정도는 TCDD을 단독투여한 흰쥐 간의 무게에 비하여 약간 감소하였다. TCDD를 단독투여한 흰쥐에서의 신장(Kidney)의 무게는 대조군에 비하여 다소 감소하였으나 통계적으로는 유의하지 않았다. TCDD와 홍삼추출물을 병행투여한 흰쥐에서의 신장의 무게의 변화는 TCDD를 단독 투여한 흰쥐에서의 결과와 차이가 없었다. 홍삼추출물 단독 투여 군에서의 신장의 무게는 실험초기(1-2일)에 대조군에 비하여 다소 감소하는 경향을 보였으나 5일 째부터는 대조군과 같은 수준으로 회복되었다. Spleen은 TCDD의 단독투여에 의해 2-3일 이내에 일시적인 감소가 있었으나 노출기간이 증가할수록 대조군 수준으로 회복되었다. 홍삼추출물단독 투여군과 TCDD와 홍삼추출물의 병행투여군에서 spleen의 무게는 대조 군에 비하여 투여 후 16일 이후에는 유의적으로 증가하였다. TCDD와 홍삼추출불의 단독 또는 병행 투여군에서의 뇌의 무게는 유의적인 변화를 보이지 않았다. 실험동물 뇨에서의 specific gravity는 대조군에서 주령에 상관없이 대체적으로 1.030 이상의 수준을 유지하였으나 홍삼 추출물을 단독 투여한 흰쥐에서는 투여 후 14일부터 specific gravity가 1.02 수준으로 낮아지는 경향이 나타났다. TCDD 단독 투여군에서는 투여 초기에 specific gravity가 1.02 수준으로 감소하는 경향이 있었으나 홍삼추출물을 병행투여했을 때 1.02 수준으로 감소하는 경향이 14일 이후에 나타났다. 실험동물 뇨에서의 Total protein 함량은 대조군에서 전체 실험 기간 동안에 100 μg/dL 수준을 유지하였으나, TCDD 단독 투여군과 TCDD와 홍삼추출물의 병행 투여군에서는 300μg/dL 이상의 함량을 나타내는 개체수가 증가하는 현상을 보였다. 한편, 홍삼추출물 단독 투여군에서는 대조군에서와 비슷한 Total protein 함량의 수준을 나타내었다. 뇨에서 ketone body 의 함량은 대조군에서 주령의 증기에 따라 높아지는 경향을 나타내었으나 실험군 간의 차이는 나타나지 않았다. Glucose, ketone, bilirubin, Occult blood, nitrite 및 urobilinogen의 함량은 모든 실험군에서 거의 유사하게 나타났으며, pH 값은 주령의 증가에 따라 높아지는 경향이 특징적이었으나 실험군 간의 차이는 나타나지 않았다. 혈액화학적 검사결과 TCDD의 단독투여에 의한 AST는 대조군에 비하여 전 실험기간에 걸쳐서 전반적으로 높게 나타났으며, 특히 32일 실험군에서 가장 높은 AST 값을 나타내었다. 홍삼추출물의 단독 투여에 의한 AST는 TCDD의 단독 투여군과는 대조적으로 오히려 노출기간이 경과할수록 감소하였다. 그리고 TCDD의 투여에 의해 증가된 AST는 홍삼추 출물을 병행투여 한지 16일부터 정상 수준으로 회복되었다. TCDD를 단독 투여한 흰쥐 혈청 ALT의 활성은 16일 까지는 대조군의 ALT 활성과 비슷한 수준이었으나 32일 째에는 대조군에 비하여 상당히 증가하였다. 이에 비하여 TCDD와 홍삼추출물을 병행 투여한 실험군에서는 16일군과 32일군에서 ALT의 활성이 급격히 감소하여 대조군의 ALT 활성보다 낮게 나타났다. 홍삼을단독 투여한 실험군에서의 ALT 활성은 전 실험기간동안에 ALT 활성에 영향을 주지 않았다. To achieve a better understanding of protective effects of water extracts of Panax ginseng against TCDD-induced toxicities, we monitored physiological and clinical changes in rat for 4 weeks after administrations of each Panax Ginseng extract or TCDD, and co-administration of the two materials. For this study, 120 male Sprague-Dawley (SD) rats weighing 190-210 g each (8 weeks old) were divided into four groups: TCDD-administered, co-administered group with TCDD and ginseng extract, ginseng extract-administered, and control group. The TCDD-administered group received single dose of TCDD in a corn oil vehicle (25 μg/kg body weight) by intraperitoneal administration on Day 1. The Panax ginseng extracts-administered group received intraperitoneally 100 mg/kg body weight every other day for one month. For the co-administered group with TCDD and ginseng extracts Panax ginseng extracts were intrapelitoneally administered to rats at 100 mg/kg body weight every other day for one month after a single intrapelitoneal dose of 25 μg of TCDD/kg body weight on Day 1. Panax ginseng extracts attenuated the mortality induced by TCDD administration. The extracts also slightly attenuated the TCDD-induced body weight loss. Administration of TCDD alone increased liver weight at 2, 5, and 16 days after administration of TCDD. Administration of Panax ginseng extracts rather decreased liver weight through whole the experimental period, but which was statistically insignificant. Administration of TCDD alone at 25 μg/kg body weight increased both serum enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 32 days, indicating that liver damage occun'ed maximally at that time. Ginseng extract administration caused insignificant changes in serum ALT, but gradually decreased in AST as the exposure time increased. Coadministration of TCDD and ginseng extracts caused serum AST activity to significant recovery to nonnal value at 16 days and 32 days after exposure to TCDD. The extracts also signiticantly decreased the TCDD-induced ALT activity after 16 days of TCDD administration. These results suggest that Panax ginseng extracts may possess a protective effect against TCDD-induced toxicities including hepatotoxicity in rats.