Cytochrome P-450 유도물질을 처리한 흰쥐 간의 마이크로좀 Aryl Hydrocarbon Hydroxylase 활성도와 혈액의 Polyamines 농도와의 관계

박기현,이윤형,Park, Ki-Hyun,Lee, Youn-Hyung 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

Cytochrome P-450 (P-450) 유도물질인 phenobarbital (Pb)과 3-methylcholanthrene (3-MC)을 흰쥐 (S.D., male, 150-160 g)에 처리한 후 간 마이 크로좀의 P-450 유도에 따른 Aryl Hydrocarbon Hydroxylase (AHHase) 활성도와 혈 액중의 polyamines 분포 및 농도와의 관계를 검토하였다. P-450-b 유도물질인 Pb 처리는 P-450 함량을 상당히 증가시키면서 대조구의 polyamines 분포 (putrescine, spermidine, spermine)와는 달리 spermidine, spermine의 분포를 나타내지만 총 polyamines 농도는 차이가 없었다. P-450-c 유도물질이며 강력한 발암물질인 3-MC 처리는 putrescine, spermidine의 분포로서 특히 putrescine은 대조구의 10배로, 그리고 총 polyamines 농도는 2배 증가되었으며 AHHase 활성도는 12배 이상 증가폭을 보였다. 이와 같은 현상은 putrescine 생합성에 관여하는 ornithine decarboxylase가 3-MC의 대사산물에 의해 그 활성이 촉진되어 비정상적으로 putrescine이 축적되며 polyamine oxidase가 spermine의 분해를 증가시킨 것으로 P-450-c의 유도에 따른 AHHase 활성도 증가와 polyamines의 생합성 또는 분해와는 밀접한 관계가 있는 것으로 사료된다. The relationship between polyamines in blood and aryl hydrocarbon hydroxylase (AHHase, EC, 1.14.14.1) activities in hepatic microsomes was observed when rats (Sprague - Dawley, male, 150-160 g) were treated intraperitoneally with phenobarbital (Pb) or 3-methylcholanthrene (3-MC) as potent inducers of cytochrome P-450 (P-450) isoenzymes. In the case of Pb-treatment for an induction of P-450-b, P-450 contents of hepatic microsomes were significantly enhanced and total polyamine levels in blood had no change After administration of 3-MC as a carcinogen and an inducer of P-450-c, the major polyamines measured in blood were putrescine and spermidine and total polyamine level was increased over 2-fold. In particular, putrescine concentration was enhanced drastically when compared with control value (10-fold). These results indicate that only the putrescine was accumulated abnormally because ornithine decarboxylase and polyamine oxidase as enzymes of polyamine synthesis or degradation were stimulated by metabolites of 3-MC treated. On the other hand, hepatic microsomal AHHase activities by the induction of P-450-c were increased over 13-fold. In this respect, a relationship is suggested between AHHase activities enhanced from induction of P-450-c and synthesis or degradation of polyamines.

Differential Inhibition of Liver Microsomal Cytochrome P-450-Dependent Monooxygenases by Hesperetin

문자영,이윤형,박기현,Mun, Ja-Yeong,Lee, Youn-Hyung,Park, Ki-Hyun 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3

Cytochrome P-450-dependent monooxygenase (P-450-MO)는 스테로이드 등의 대사에 도 관여하지만 이물질 특히 환경오염물질 등의 생체내 활성화 대사를 촉진한다. 또한 P-450은 여러 동위 효소로 구성되어 있으며 각각의 기질 특이성을 나타낸다. 따라서 동위 효소 유도 물질로 phenobarbital (PB)과 3-methylcholanthrene (MC)을 흰쥐 (SP-D, male, 150-160g)에 각각 처리하여 간의 microsomes (mis)에 존재하는 P-450을 스펙트럼 차이로 부터 그리고 단일항체와 $^{35}S$-rabbit antimouse IgG를 사용한 방사면역학적인 방법에 의해 P-450-b (P-450) type과 P-450-c (P-448) type이 주된 동위 효소로 각각의 mis에 존재하는 것을 확인하였다. 이와 같은 mis에 hesperetin (2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one)을 첨가한 스펙트럼 특성은 동일한 modified type II (Peak, 415 nm; trough, 385 nm)를 나타내므로서 hesperetin이 MO에 대한 억제효과가 있음을 알았다. hesperetin의 benzo(a)pyrene hydroxylase (BPH), 7-ethoxycoumarin O-deethylase (ECDE), aniline hydroxylase (AH)에 대한 inhibitory potency ($I_{50}$)는 PB-mis 또는 MC-mis, 즉, P-450의 동위 효소에 따라 다소 차이는 있으나 측정된 MO 들의 활성도에 상관 없이 비교적 강력한 것으로 나타났다. 한편 MO에 대한 hesperetin의 inhibition type과 inhibition constant ($K_i$)를 측정한 결과 PB-mis-BPH에 대하여 noncompetitive inhibitor로 작용했으며 이때의 $K_i$ 값은 $5.42\;{\mu}M$ 이었다. 그리고 MC-mis-BPH에는 mixed inhibitor로 $K_i$ 값은 $14.5\;{\mu}M$로 전자보다 높았다. 또한 MC-mis-ECDE에는 hesperetin이 mixed inhibition을 나타냈으며 $K_i$ 값은 $1.67\;{\mu}M$로 competitive inhibition을 나타내는 PB-mis-ECDE의 $18\;{\mu}M$보다 상당히 낮았을 뿐 아니라 BPH의 값들보다도 훨씬 낮은 경향이었다. PB-mis-와 MC-mis-AH에는 hesperetin이 같은 mixed inhibitor로 작용했으며 $K_i$ 값은 각각 20.5, $34\;{\mu}M$로 높은 수치를 보였다. 따라서 hesperetin의 MO에 대한 선택적이며 차별적인 억제효과는 측정되는 mis에 따라 그리고 MO에 따라 상당한 차이를 보이고 있으며 특히 MC-microsomal P-450-dependent ECDE에 가장 강력한 억제효과를 나타냈다. The effect of hesperetin (2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-One) on the inhibition of the liver microsomal monooxygenases was investigated from the male Sprague Dawley rats (150-160 g) pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) in vitro. The major form of PB- or MC-induced P-450 isozyme was identified from characteristic CO-difference spectra (450 nm and 448 nm) and from results by using the specific monoclonal antibodies of P-450 isozymes and the secondary antibody ($^{35}S$-rabbit antimouse IgG), namely the major form of PB-microsomal P-450 was P-450-b type and that of MC-microsomal-450 was P-450-c (P-448) type. A very similar spectral changes of the modified type II (peak: 415 nm, trough: 385 nm) were caused from the PB- and MC-aerobic liver micro somes added with hesperetin, respectively. From the result of this spectral changes and of the high inhibitory potency ($I_{50}=17-75\;{\mu}M$), we found that this compound is an effective inhibitor towards of the oxidative reactions of microsomal monooxygenases tested, namely benzo(a)pyrene hydroxylase (BPH), 7-ethoxycoumarin o-deethylase (ECDE) and aniline hydroxylase (AH). Hesperetin exerted a mode of noncompetitive inhibition on the BPH of PB-microsomes and of mixed inhibition on the same enzyme of MC-microsomes with inhibition constant ($K_i$) of 5.42 and $14.5\;{\mu}M$, respectively. On the other hand, the hesperetin inhibited competitively the ECDE of PB-microsomes and its $K_i$ value was $18\;{\mu}M$, whereas the ECDE of MC-microsomes was markedly inhibited as a mixed mode by this compound and the $K_i$ value was remarkably low ($1.67\;{\mu}M$). In the case of the AH of PB and MC-microsomes, the activities exhibited the same mode of mixed inhibition and the $K_i$ values in general were higher than those of BPH or ECDE. From this observations hesperetin clearly demonstrated that it is an inhibitor of the oxidative metabolism of exogenous substances catalyzed by the liver microsomal P-450-dependent monooxygenases. In particular, this compound indicated that it is a new potent inhibitor of ECDE from results of the high affinity for microsomes containing a great proportion of P-450-c in total P-450 content. We also found that the differences exist between the mechanisms of inhibition according to the form of P-450 isozyme present in microsomes of differently pretreated rats and/or according to the monooxygenase tested.

Cytochrome P - 450 유도물질 처리한 흰쥐 간의 마이크로좀 Aryl Hydrocarbon Hydroxylase 활성도와 혈액의 Polyamines 농도와의 관계

박기현,이윤형 ( Ki Hyun Park,Youn Hyung Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

The relationship between polyamines in blood and aryl hydrocarbon hydroxylase (AHHase, EC, 1.14.14.1) activities in hepatic microsomes was observed when rats (Sprague - Dawley, male, 150 160 g) were treated intraperitoneally with phenobarbital (Pb) or 3 - methylcholanthrene (3 - MC) as potent inducers of cytochrome P - 450 (P - 450) isoenzymes. In the case of Pb - treatment for an induction of P - 450 - b, P - 450 contents of hepatic microsomes were significantly enhanced and total polyamine levels in blood had no change After administration of 3 - MC as a carcinogen and an inducer of P - 450 - c, the major polyamines measured in blood were putrescine and spermidine and total polyamine level was increased over 2 - fold. In particular, putrescine concentration was enhanced drastically when compared with control value (10 - fold). These results indicate that only the putrescine was accumulated abnormally because ornithine decarboxylase and polyamine oxidase as enzymes of polyamine synthesis or degradation were stimulated by metabolites of 3 - MC treated. On the other hand, hepatic microsomal AHHase activities by the induction of P - 450 - c were increased over 13 - fold. In this respect, a relationship is suggested between AHHase activities enhanced from induction of P - 450 - c and synthesis or degradation of polyamines.

흰쥐 간의 Cytochrome P - 450 - Dependent Monooxygenase 에 대한 Hesperetin 의 억제효과

박기현,이윤형,문자영 생화학분자생물학회 1991 BMB Reports Vol.19 No.3

The effect of hesperetin (2,3-dihydro-5,7-dihydroxy-2-(3-hvdroxy-4-methoxyphenyl)-4H-1-benzopyran-4-One) on the inhibition of the liver microsomal monooxygenases was investigated from the male Sprague Dawley rats (150-160 g) pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) in vitro. The mayor form of PB- or MC-induced P-450 isozyme was identified from characteristic CO-difference spectra (450 ㎚ and 448 ㎚) and from results by using the specific monoclonal antibodies of P-450 isozymes and the secondary antibody (^(35)S-rabbit antimouse IgG), namely the major form of PB-microsomal P-450 was P-450-b type and that of MC-microsomal P-450 was P-450-c (P-448) type. A very similar spectral changes of the modified type II (peak: 415 nm, trough: 385 nm) were caused from the PB- and MC-aerobic liver microsomes added with hesperetin, respectively. From the result of this spectral changes and of the high inhibitory potency (I_(50)=17-75 μM), we found that this compound is an effective inhibitor towards of the oxidative reactions of microsomal monooxygenases tested, namely benzo(a)pyrene hydroxylase (BPH), 7-ethoxycoumarin ο-deethylase (ECDE) and aniline hydroxylase (AH). Hesperetin exerted a mode of noncompetitive inhibition on the BPH of PB-microsomes and of mixed inhibition on the same enzyme of MC-microsomes with inhibition constant (K_i) of 5.42 and 14.5 μM, respectively. On the other hand, the hesperetin inhibited competitively the ECDE of PB-microsomes and its K_i value was 18 μM, whereas the ECDE of MC-microsomes was markedly inhibited as a mixed mode by this compound and the K value was remarkably low (1.67 μM). In the case of the AH of PB and MC-microsomes, the activities exhibited the same mode of mixed inhibition and the K_i values in general were higher than those of BPH or ECDE. From this observations hesperetin clearly demonstrated that it is an inhibitor of the oxidative metabolism of exogenous substances catalyzed by the liver microsomal P-450-dependent monooxygenases. In particular, this compound indicated that it is a new potent inhibitor of ECDE from results of the high affinity for microsomes containing a great proportion of P-450-c in total P-450 content. We also found that the differences exist between the mechanisms of inhibition according to the form of P-450 isozyme present in microsomes of differently pretreated rats and/or according to the monooxygenase tested.