HNFs에 의한 CYP1B1 유전자 발현 조절 및 소핵시험법을 이용한 유전독성 평가법

Abstract

Part 1 The mechanism of liver-specific expression of P450 gene is unique. It is widely known that expressions of liver specific genes, such as albumin and C/EBP are regulated by interaction with liver-enriched transcription factor (LETF) at the transcriptional level. Among LETFs of increasingly considerable importance, hepatic nuclear factors (HNFs), in particular, are reported to be involved in the regulation of gene expression as an important factor in a few of significant liver specific genes. In an effort to find out a relationship between each nuclear receptor of HNFs and CYP1B1 gene expression, and ultimately understand the mechanism of the regulation of CYP1B1 gene expression, 5'-flanking DNA of human CYP1B1 was cloned into vector containing luciferase reporter genes (phCYP1B1-luc), and then transfected with pHNFs into Hepa-I cells. And luciferase activity expressed following treatment of various chemicals was measured. After transfection with 100 ng phCYP1B1-luc along with 20, 40, 60, 80, 100, 120 and 140 ng HNF1α, cells were treated with vehicle (0.1% DMSO) or TCDD. When cells containing HNF1α were treated with vehicle, the luciferase activities were 2.87-, 6.05-, 6.23-, 7.97-, 9.95-, 4.00- and 1.47-fold, respectively. When cells containing HNF1α were treated with 10-9 M TCDD, the luciferase activities were 8.62-, 118.57-, 142.42-, 147.31-, 165.10-, 186.29- and 168.86-fold, respectively. When cells containing HNF1α were treated with 10-11 M TCDD, the luciferase activities were 14.78-, 23.99-, 27.58-, 30.77-, 35.04-, 35.27- and 20.73-fold, respectively. The luciferase activity was increased as increased the amount of HNF1α. The luciferase activity in TCDD-treated group is significantly higher than the luciferase activity in vehicle group. After transfection with 100 ng phCYP1B1-luc along with 20, 40, 60, 80, 100, 120 and 140 ng HNF1β, cells were treated with vehicle (0.1% DMSO) or TCDD. When cells containing HNF1β were treated with vehicle, the luciferase activities were 2.96-, 5.74-, 6.38-, 8.54-, 9.23-, 2.61- and 1.63-fold, respectively. When cells containing HNF1β were treated with 10-9 M TCDD, the luciferase activities were 6.03-, 133.97-, 150.00-, 129.88-, 116.19-, 109.93- and 119.52-fold, respectively. When cells containing HNF1β were treated with 10-11 M TCDD, the luciferase activities were 8.43-, 56.08-, 58.62-, 58.26-, 50.60-, 51.36- and 36.34-fold, respectively. The luciferase activity was increased as increased the amount of HNF1β. The luciferase activity in TCDD-treated group is significantly higher than the luciferase activity in vehicle group. After transfection with 100 ng phCYP1B1-luc along with 20, 40, 60, 80, 100, 120 and 140 ng HNF3β, cells were treated with vehicle (0.1% DMSO) or TCDD. When cells containing HNF3β were treated with vehicle, the luciferase activities were 1.16-, 1.88-, 2.10-, 2.93-, 3.31-, 3.63- and 1.90-fold, respectively. When cells containing HNF3β were treated with 10-9 M TCDD, the luciferase activities were 4.20-, 5.50-, 5.06-, 5.15-, 5.34-, 6.00- and 6.63-fold, respectively. When cells containing HNF3β were treated with 10-11 M TCDD, the luciferase activities were 5.48-, 7.34-, 8.50-, 11.65-, 14.05-, 13.66- and 10.35-fold, respectively. The luciferase activity was increased as increased the amount of HNF3β. After transfection with 100 ng phCYP1B1-luc along with 20, 40, 60, 80, 100, 120 and 140 ng HNF4α, cells were treated with vehicle (0.1% DMSO) or TCDD. When cells containing HNF4α were treated with vehicle, the luciferase activities were 3.29-, 4.28-, 2.88-, 2.32-, 2.13-, 0.69- and 0.37-fold, respectively. When cells containing HNF4α were treated with 10-9 M TCDD, the luciferase activities were 27.81-, 29.23-, 26.65-, 17.71-, 13.20-, 11.86- and 9.75-fold, respectively. When cells containing HNF4α were treated with 10-11 M TCDD, the luciferase activities were 14.32-, 26.49-, 33.15-, 42.27-, 39.24-, 38.08- and 16.38-fold, respectively. The luciferase activity was increased as increased the amount of HNF4α. The luciferase activity in TCDD-treated group is higher than the luciferase activity in vehicle group. CYP1B1 enzyme transforms estradiol into 4 hydroxyestradiol, a carcinogenic metabolite. 1 nM TCDD increased the luciferase activity by 20-fold. The concomitant treatment of TCDD with 17β-estradiol decreased TCDD-induced luciferase activity in a estrogen concentration manner. The inducibility of TCDD on the luciferase activity was mostly recovered with concomitant tamoxifen. Diethlystilbestrol (DES), a synthetic estrogen, did not affect TCDD-induced luciferase activity. Cobalt chloride, a substance inducing hypoxia, also inhibited the increase of luciferase activity. The experiment of flavonoids like chrysin, daidzein, genistein, morin, and naringenin showed that flavonoids inhibited TCDD-induced luciferase activity at low concentration, but morinand naringenin increased the luciferase activity at high concentration. However, the transfection of pHNF1β reduced TCDD-induced luciferase activity in a dose dependent manner Part II The Micronucleus assay that evaluates genotoxicity of various chemicals by using micronucleus, small membrane-bound DNA fragments generated from derivative or disorder of DNA or chromosomal level, as a biomarker was suggested. This study used a cytokinesis-block micronucleus assay that uses micronucleus counted in a cell completely separated from main nucleus by adding cytochalasin B, an actin inhibitor. This experiment could detect clastogen, a substance that induces chromosomal damage, and aneugen, a substance that induces aneuploidy in the case of mitosis and meiosis. In vitro micronucleus assay using various cell types is widely used, as it is effective in evaluating genotoxicity with a simple way of counting micronuclei, and it is under the examination of OECD. This study sought to validate a cytokinesis-block micronucleus assay and find out a correlation between genotoxicity and carcinogenecity. A total of 8 substances were taken for the test to validate the propriety of genotoxicity. Among them are two kinds of genotoxic carcinogen (1,2-dibromoethane and glycidol), two kinds of genotoxic noncarcinogen (8-hydroxyquinoline and emodin), two kinds of nongenotoxic carcinogen (methycarbamate and o-nitrotoluene), and two kinds of non -genotoxic noncarcinogen (D-mannitol and 1,2 dichlorobenzene). In an effort to examine whether the substances show genotoxicity by inducing damage on a chromosomal level, CHO-K1 (Chinese Hamster Ovary) cells suggested in the OECD guideline was used as cell line for an in vitro micronucleus assay. The test compound was treated without S9 or with S9 for 4 hours after cytokinesis was inhibited by treating cytochalasin B in a CHO-K1 cell. Micronuclei were observed after the process of dying acridine orange and fixing methanol. Positive control was included in order to improve reliability of the test and the same test was conducted three times to evaluate reproducibility. Micronucleus frequency was decided to be positive when it grows dose-dependently and statistically significantly. Cyclophosphamide, positive control substance, increased the formation of micronuclei with S9 concen-tration-dependently at a concentration of 2.5, 5, 10 ㎍/㎖, compared with a negative control, which shows that this experiment was properly conducted. There was not a significant increase without S9, compared with a negative control. In the case of 1,2-dibromoethane and glycidol, micronuclei increased concentration-dependently with or without S9, compared with a negative control. O-nitrotoluene, 1,2-dichlorobenzene, 8 hydroxyquinoline, emodin, methyl carbamate and D-mannitol did not show a significant increase with or without S9, compared with a negative control. The above mentioned results decided that 1,2-dibromoehtnae and glycidol are micronuclei frequency positive, and 8-hydroxyquinoline, emodin, methyl carbamate, o-nitrotoluene, D-mannitol, 1,2-dichlorobenzene are micronuclei frequency negative.;Part 1 P450의 간 특이적인 발현기전은 꽤 독특하다. 알부민(albumin) 같은 간 특이적 유전자의 발현은 전사 단계에서 liver-enriched transcription factor (LETF)와의 상호작용에 의해 조절되는 것이 이미 알려져 있다. 최근 그 중요성이 대두되고 있는 LETF 가운데서도 특히 hepatic nuclear factors (HNFs)는 몇몇 간 특이적 유전자에서 중요한 요소로서 유전자 발현 조절에 관여하고 있다는 보고가 있었다. CYP1B1 유전자 조절 기전의 이해를 돕기 위하여 이러한 HNFs 등의 핵 내 수용체와 CYP1B1 유전자 발현 사이의 상관성을 알아보고자 사람의 CYP1B1 5'-flanking DNA를 luciferase reporter 유전자를 포함한 vector에 clone하여 Hepa-I 세포에 pHNFs와 cotransfection 한 뒤, 여러 가지 화합물을 처리하여 발현되는 luciferase 활성을 측정하였다. Hepa-I 세포에 hCYP1B1-Luc 100 ng 과 HNF1α, 1β, 3β, 4α 를 농도별로 contransfection한 뒤, 10-9 M TCDD를 처치하고 24시간 뒤 luciferase 활성을 측정하여 0.1% DMSO 대조군에 대한 luciferase 활성 증가를 관찰했다. hCYP1B1 -Luc 100 ng 과 HNF1α를 0, 20, 40, 60, 80, 100, 120, 140 ng 씩 각각 cotransfection한 뒤, 0.1% DMSO를 처치한 경우 2.87, 6.05, 6.23, 7.97, 9.95, 4.00 1.47 배, 10-9 M TCDD를 처치한 경우 48.62, 118.572, 142.42, 147.31, 165.10, 186.29, 168.86 배, 10-11 M TCDD를 처치한 경우 14.78, 23.99, 27.58, 30.77, 35.04, 35.27, 20.73 배 증가하였다. HNF1α 농도가 증가함에 따라 활성이 증가하는 경향을 보인다. TCDD를 처치한 경우 0.1% DMSO 처치군과 그 경향과 비슷하나 활성정도는 매우 높게 나타난다. hCYP1B1-Luc 100 ng 과 HNF1β를 0, 20, 40, 60, 80, 100, 120, 140 ng 씩 각각 cotransfection한 뒤, 0.1% DMSO를 처치한 경우 2.96, 5.74, 6.38, 8.54, 9.23, 2.61, 1.63 배, 10-9 M TCDD를 처치한 경우 96.03, 133.97, 150.00, 129.88, 116.19, 109.93, 119.52 배, 10-11 M TCDD를 처치한 경우 28.43, 56.08, 58.62, 58.26, 50.60, 51.36, 36.34 배 증가하였다. HNF1β 농도가 증가함에 따라 활성이 증가하는 경향을 보인다. TCDD를 처치한 경우 0.1% DMSO 처치군과 그 경향과 비슷하나 활성정도는 매우 높게 나타난다. hCYP1B1-Luc 100 ng 과 HNF3β를 0, 20, 40, 60, 80, 100, 120, 140 ng 씩 각각 cotransfection한 뒤, 0.1% DMSO를 처치한 경우 1.16, 1.88, 2.10, 2.93, 3.31, 3.63, 1.90 배, 10-9 M TCDD를 처치한 경우 4.20, 5.50, 5.06, 5.15, 5.34, 6.00, 6.63 배, 10-11 M TCDD를 처치한 경우 5.48, 7.34, 8.50, 11.65, 14.05, 13.66, 10.35 배 증가하였다. HNF3β 농도가 증가함에 따라 활성이 증가하는 경향을 보이나 다른 HNFs의 활성에 비하여 크게 증가하지 않는다. TCDD를 처치한 경우 0.1% DMSO 처치군과 그 경향과 비슷하다. hCYP1B1-Luc 100 ng 과 HNF4α를 0, 20, 40, 60, 80, 100, 120, 140 ng 씩 각각 cotransfection한 뒤, 0.1% DMSO를 처치한 경우 3.29, 4.28, 2.88, 2.32, 2.13, 0.69, 0.37 배, 10-9 M TCDD를 처치한 경우 27.81, 29.23, 26.65, 17.71, 13.20, 11.86, 9.75 배, 10-11 M TCDD를 처치한 경우 14.32, 26.49, 33.15, 42.27, 39.24, 38.08, 16.38 배 증가하였다. HNF4α 농도가 증가함에 따라 활성이 증가하는 경향을 보인다. TCDD를 처치한 경우 0.1% DMSO 처치군과 그 경향과 비슷하나 활성정도는 높게 나타난다. 17β-estradiol을 TCDD와 병용 처치한 경우, TCDD에 의해 유도 발현된 luciferase 활성을 농도 의존적으로 감소시켰다. 그리고 pHNFs를 cotransfection 한 경우에도 TCDD에 의해 유도 발현된 luciferase 활성을 감소시켰다. 그러나 이러한 감소 효과는 ER의 부분 길항제인 tamoxifen과의 병용 처치로 대부분 회복되었다. 합성여성호르몬의 일종인 diethlystilbestrol (DES)은 TCDD에 의해 유도된 luciferase 활성에 별다른 영향을 보이지 않았다. 하지만 pHNFs를 cotransfection한 경우에는 TCDD에 의해 유도 발현된 luciferase 활성을 감소시켰다. 저산소 상태 유도 물질인 cobalt chloride 또한 TCDD에 의한 luciferase 활성증가를 억제하였다. Chrysin, daidzein, genistein, morin, naringenin 같은 flavonoids에 대해서도 실험한 결과 이러한 flavonoids는 저농도에서 TCDD에 의해 유도된 luciferase 활성을 저해하지만, 고농도에서는 morin과 naringenin의 경우 오히려 luciferase 활성을 증가시켰다. 하지만 pHNFs를 cotransfection 한 경우 농도 의존적으로 TCDD에 의해 유도된 luciferase 활성을 감소시켰다. Part II DNA 또는 염색체 수준의 장애나 부가 생성물의 결과로 생성된 작은 핵조각 (small membrane-bound DNA fragments)인 소핵을 유전독성 생체 지표 (biomarker)로 하여 다양한 물질들의 유전독성을 평가하는 소핵시험법이 제안되었다. 본 연구에서는 다양한 소핵시험법 중 액틴 저해제(atin inhibitor)인 cytochalasin B를 첨가하여 주핵이 완전히 분리된 세포에서 소핵을 세는 세포질분열억제 소핵시험법(cytokinesis-block micronucleus assay)을 이용하였다. 이 시험법을 통하여 염색체 손상을 유발하는 성질을 가진 물질, 즉 clastogen이나 유사분열이나 감수분열시에 염색체수의 변화(aneuploidy)를 유발하는 물질, 즉 aneugen 을 검출할 수 있다. 다양한 세포 타입을 이용한 생체 외 소핵시험법은 소핵을 계수하는 방법이 간단하여 유전독성을 평가하는데 효과적인 방법이라 일반적으로 널리 쓰여지고 있으며 현재 OECD에서 검토 중에 있는 시험법이다. 본 연구에서는 세포질분열억제 소핵시험법을 검증하고 유전독성(genotoxicity)과 발암성(carcinogenecity)의 상관관계에 관하여 알아보고자 하였다. 2종의 유전독성 발암물질(genotoxic carcinogen)로서 1,2-dibromoethane과 glycidol, 2종의 유전독 성 비발암물질(genotoxic noncarcinogen)로서 8 hydroxyquinoline과 emodin, 2조의 비유전독성 발암물질(nongenotoxic carcinogen)로서 methycarbamate와 o-nitrotoluene, 2종의 비유전독성 비발암물질(nongenotoxic noncarcinogen)로서 D-mannitol과 1,2-dichlorobenzene, 총 8종의 시험물질을 대상으로 유전독성시험법의 타당성을 검증하였다. 시험물질들이 염색체 수준에서의 손상을 유도하여 유전독성을 나타내는지를 측정하기 위하여 시험계는 OECD 가이드라인에 제시되어 있는 CHO-K1 (Chinese Hamster Ovary) 세포를 이용하여 생체외 소핵시험 (in vitro micronucleus assay)을 수행하였다. CHO-K1 세포에 cytochalasin B를 처리하여 세포질분열을 저해시킨 후, S9 혼합액 부재하(직접법) 또는 존재하(대사활성화법)에서 시험물질을 4시간 처리하였다. Methanol 고정 및 acridine orange 염색과정을 거친 후에 소핵을 관찰하였다. 시험의 신뢰성을 위해 양성대조군을 포함하고, 재현성 평가를 위해 3 회 반복 시험을 수행하였다. 소핵시험의 양성 판정은 소핵유발정도가 용량 의존적인 증가반응을 보이며 통계학적으로 유의적인 경우 양성으로 판정하였다. 양성대조물질인 cyclophosphamide는 S9 혼합액 존재하에서, 음성대조군에 비해 2.5, 5, 10 ㎍/㎖의 농도에서 농도 의존적으로 소핵형성을 증가시켜 본 실험이 적절히 행하여졌음이 확인되었다. S9 혼합액을 처리하지 않은 경우는 음성대조군에 비해 유의성 있는 증가를 나타내지 않았다. 시험물질인 1,2-dibromoethane, glycidol은 S9 혼합액 존재하와 부재하 모두에서 음성대조군에 비해 농도 의존적으로 소핵이 증가하였다. O-nitrotoluene, 1,2-dichlorobenzene, 8 hydroxyquinoline, emodin, methyl carbamate, D-mannitol은 S9 혼합액 존재하와 부재하 모두에서 음성대조군에 비해 유의성 있는 증가를 나타내지 않았다. 이상의 결과에서 1,2-dibromo -ehtnae, glycidol은 소핵 유발능 양성, 8-hydroxyquinoline, emodin, methyl carbamate, o-nitrotoluene, D-mannitol, 1,2-dichlorobenzene는 소핵 유발능 음성 으로 판정하였다. 발암물질에 소핵유발능 양성을 나타낸 민감도는 50%로 나타났고, 비발암물질에 소핵유발능 음성을 나타낸 정확도는 100%로 나타났다.