랫트에서 Paraquat 급성투여에 의한 간내 황함유아미노산 대사의 변화 : Alterations in Transsulfuration Reactions in Rat Liver Induced by Acute Administration of Paraquat

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서울대학교 대학원
학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2012. 2. 김영철.
본 연구에서는 체내 항산화방어에 중요한 역할을 하는 간내 황함유아미노산 대사에 oxidant 와 antioxidant 가 주는 변화를 시험하였다. 랫트에 Paraquat (PQ; 20 mg/kg) 을 일회 복강 투여 후 4 시간과 24 시간대에서 지표변화를 측정하였다. Quercetin (QUE) 은 PQ 투여 후 2 시간과 6 시간에 각각 50 mg/kg 의 용량으로 복강 투여하였다.
PQ 은 24 시간에서 간내 malondialdehyde (MDA) 함량을 유의적으로 증가시켰으며, 이는 QUE 에 의해 더욱 증폭되었다. Peroxyl radical, hydroxyl radical, peroxynitrite 에 대한 간내 총산소라디칼 제거능력 (TOSC) 은 PQ 단독투여시 감소되는 경향을 보였으며 QUE 병용투여군에서 뚜렷한 감소를 보였다. 이와 달리 혈액에서는 perxoxyl radical 과 peroxynitrite 의 총산소라디칼 제거능력이 QUE 병용투여에 의해 증가되었으며 PQ 단독투여에 의해서는 유의적인 변화가 관찰되지 않았다. 한편, 간내 methionine 은 큰 차이를 보이지 않았으나, S-adenosylmethionine (SAM) 은 대조군 대비 모든 투여군에서 1.2 배가량 증가하였다. S-adenosylhomocysteine (SAH) 은 PQ 단독투여하였을 때 유의적으로 증가하였다. Methionine 으로부터 SAM 으로의 대사를 매개하는 methionine adenosyltransferase (MAT) 활성은 PQ 단독투여 24 시간대와 QUE 병용투여 그룹에서 감소하였다. Homocysteine은 변화하지 않았으나 homocysteine 에서 methionine 으로 변환시키는 betaine-homocysteine methyltrasncferase (BHMT) 활성은 PQ 단독투여 24 시간대에서 감소하며, homocysteine 을 cystathionine 으로 대사시키는 cystathionine β-synthethase (CBS) 활성이 PQ 단독투여 24 시간대와 QUE 병용투여 그룹에서 증가하고 cystathionine도 PQ 단독투여와 QUE 병용투여 모두에서 증가하였다. Cystathionine γ-lyase (CγL) 활성은 유의적인 변화가 없었다. Cysteine 은 PQ 단독투여와 QUE 병용투여 모두에서 유의적으로 증가하였다. Cysteine의 catabolism 에 관여하는 cysteine dioxygenase (CDO) 활성은 유의적인 차이가 없었으며, 중간생성물인 hypotaurine 은 변화가 보이지 않았으나, 최종대사물인 taurine 은 PQ 단독투여 24 시간대와 QUE 병용투여 그룹에서 증가하였다. Cysteine 에서 glutathione (GSH) 합성에 관여하는 γ-glutamylcysteine synthetase (GCS) 의 활성은 PQ 단독투여 24 시간대와 QUE 병용투여 그룹에서 감소하였으나 그 대사물인 GSH 은 유의적인 차이가 없었다.
종합하면, 본 실험은 PQ 의 급성투여는 산화적 스트레스 지표의 증가를 유도하고, 황함유아미노산 대사에 현격한 변화를 유발시킴을 보여준다. 특히 cystathionine, cysteine 을 증가시키며 GSH pathway 보다 taurine pathway 를 활성화시켰다. 또한 PQ 에 의한 MAT 활성감소에도 불구하고 SAM 은 증가하며, 이는 PQ 에 의한 SAM decarboxylase 감소와 유관한 것으로 사료된다.
Oxidative stress has recently been shown to play an important role in various diseases. Especially in alcoholic liver disease, nonalcoholic steatohepatitis, and hepatitis type C, oxidative stress play an important role in the pathophysiological changes that progress to liver cirrhosis and finally to hepatocellular carcinoma. However, the roles of oxidative stress in these liver disease are not yet fully understood, and further studies on oxidative stress in liver are required.
In the present study, the changes in hepatic metabolism of GSH and S-amino acids induced by oxidants and antioxidants is investigated. Paraquat has been widely used as a model substance to induce oxidative stress in several organs. Quercetin is known to act as an effective antioxidant by chelating transition metal ions and scavenging free radicals. The rats were injected with a single dose of paraquat (20 mg/kg, ip), and the alterations of the metabolites and the enzyme activities in transsulfuration pathway were monitored for 24 hrs. Quercetin (50 mg/kg, ip) was injected twice into the peritoneal cavity, at 2 and 6 hrs and rats were sacrificed 24 hrs after paraquat treatment.
The magnitude of oxidative stress was quantified by measuring malonylaldehyde production. Paraquat treatment resulted in a significant increase in malonylaldehyde levels and TOSC values. Contrary to expectation, quercetin adminstration increased lipid peroxidation. Paraquat did not affect the hepatic methionine level. S-Adenosylmethionine (SAM) was increased despite the reduction of methionine adenosyltransferase (MAT) activity. S- Adenosyl- homocysteine (SAH) was also increased at t = 4 hr, 24 hr. Homocysteine level was not changed. However, cystathionine elevated in all groups because betaine homocysteine methyltransferase (BHMT) activity was depressed at t = 24 hr and cystathionine β-synthase (CβS) activity elevated in all groups. Cystathionine γ-lyase (CγL) activity did not change but hepatic cysteine increased significantly. Cysteine dioxygenase, which mediates the metabolic conversion of cysteine to hypotaurine, did not show change in activity. Although level of hypotaurine did not change, taurine increased in paraquat 24 hr group and quercetin administration group. γ-glutamylcysteine synthetase activity, which mediates the synthesis of GSH, was inhibited. Total glutathione level was not changed.
In summary, acute adminstration of paraquat induces lipid peroxidation and has a sinificant influence on transsulfuration metabolism. Paraquat treatment decreases MAT activity but SAM and SAH concentration were elevated. It is considered that paraquat induces decrease of SAM decarboxylase activities and prevents consumption of SAM. An increase in CβS activity and decrease in BHMT activity enhance the catabolism of methionine to cysteine. Hepatic concentration of taurine increased significantly following paraquat administration, and GSH did not change as a result of inhibition of GCS activity. The role of taurine under oxidative stress remains to be examined.
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Master's Degree_약학과)
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