氯胺酮誘導大鼠肝臟細胞色素P450及其毒理意義

Abstract

細胞色素P450（Cytochrome P450; CYP或P450）乃是生物體進行生物轉換的重要酵素系統，它主要存在於肝臟中。P450的活性會受到一些外來物質改變，進而對毒性物質的活化造成影響；P450活性之改變亦是許多藥物間交互作用的機轉所在。 氯胺酮（ketamine）是一種靜脈麻醉劑，具有強力止痛及催眠作用，目前仍常被用於人體或動物的麻醉當中。氯胺酮會引起幻覺，因此近來也逐漸成為藥物濫用者所經常使用的藥物。在臨床上曾報告過反覆給予氯胺酮會造成後來給予的氯胺酮作用時間減短，而此種急性耐藥性（acute tolerance）的產生，被認為與氯胺酮活化藥物代謝酵素有關。本實驗即是以大鼠為動物模式探討氯胺酮對藥物代謝的主要酵素P450的作用並探討所產生之毒理意義與藥物交互作用。 首先以腹腔注射方式給予雄性大鼠一天兩次40 mg/kg氯胺酮，連續四天，結果發現肝臟微粒體的ethoxycoumarin O-deethylation (ECOD)、ethoxyresorufin O-dealkylation (EROD)、methoxyresorufin O-dealkylation (MROD)及pentoxyresorufin O-dealkylation (PROD) 等P450酵素活性皆有上升現象。進一步做劑量效應試驗，發現以腹腔注射方式給予雄性大鼠一天兩次10、20、40、80 mg/kg ketamine，連續四天，PROD活性上升有極佳劑量效應關係。而腹腔注射方式給予雄性大鼠一天兩次80 mg/kg氯胺酮，連續四天，也會增加大鼠肝臟erythromycin N-demethylation (END)、aniline hydroxylation、P450總量、cytochrome b5以及NADPH-P450 reductase活性。其他酵素如glutathione S-transferase (GST) 以及UDP-glucuronosyltransferase (UGT) 活性也有顯著上升；至於肝臟glutathione peroxidase活性則無明顯變化。其中PROD、ECOD、MROD與END活性上升的現象在四天後恢復原狀，EROD、aniline hydroxylation、GST與UGT活性在四天後仍較控制組高。接下來以西方點墨法發現肝臟微粒體CYP2B1/2蛋白量在10、20、40、80 mg/kg氯胺酮處理後分別上升5、6、10、13倍，80 mg/kg氯胺酮處理後，CYP1A1/2, CYP2E1與CYP3A 1/2蛋白量均上升2倍。RT-PCR實驗則發現80 mg/kg氯胺酮處理後CYP2B1/2 mRNA上升1.7倍。上述實驗顯示氯胺酮可誘導數種大鼠肝臟細胞色素P450酵素，其中以CYP2B最為顯著。 在ketamine造成的藥物交互作用的方面，發現氯胺酮前處理會縮短 2,6-雙異丙烷酚 (propofol) 對大鼠的麻醉時間，此一現象可被CYP2B抑制劑orphenadrine所回復， 2,6-雙異丙烷酚血中濃度在氯胺酮前處理大鼠下降較快。在離體實驗中發現氯胺酮前處理過的大鼠其肝臟微粒體可加速 2,6-雙異丙烷酚代謝及其代謝產物4-hydroxypropofol的生成，此一現象亦可被orphenadrine所回復。因此氯胺酮可加速 2,6-雙異丙烷酚代謝並減低其麻醉效果，此一現象與氯胺酮誘導CYP2B有關。 在氯胺酮造成的毒性增強方面，發現氯胺酮可增強四氯化碳以及古柯鹼的動物肝毒性。CYP2B抑制劑orphenadrine可回復氯胺酮加強古柯鹼肝毒性；但無法回復氯胺酮增強四氯化碳肝毒性。以RT-PCR偵測細胞激素變化或以三氯化釓清除肝臟巨噬細胞均無法改變此一現象。因此氯胺酮誘導CYP2B是古柯鹼毒性增強的重要機轉；至於氯胺酮增強四氯化碳肝毒性的機轉則尚無決定性的結論。 總結來說，氯胺酮可誘導大鼠肝臟細胞色素P450，其中對CYP2B的誘導作用最強，其毒理意義主要包括會加速 2,6-雙異丙烷酚的代謝，減低 2,6-雙異丙烷酚的麻醉效果，另外也會加強古柯鹼引起的肝毒性。

Cytochrome P450 (P450; CYP) plays a pivotal role in the biotransformation of a wide variety of xenobiotics. The activity of P450 is susceptible to induction or inhibition by numerous xenochemicals. Altered P450 activity is a common underlying mechanism of drug-drug interactions and can also affect the bioactivation of xenobiotic toxicity. Ketamine is an intravenous anesthetic that can produce potent analgesic effect and hypnosis simultaneously. In addition, ketamine can cause a trance-like state of mind in humans and consequently has become an abused substance. The development of acute ketamine tolerance is suggested to result from P450 induction by the drug. The aim of the thesis is to investigate the inductive effect of ketamine on rat hepatic P450 and explore its toxicological implications. Firstly, intraperitoneal (ip) administration of 40 mg/kg ketamine to male Wistar rats twice daily for four days was found to increase the levels of hepatic microsomal ethoxycoumarin O-deethylation (ECOD), ethoxyresorufin O-dealkylation (EROD), methoxyresorufin O-dealkylation (MROD) and pentoxyresorufin O-dealkylation (PROD). Further experiments revealed a strong dose-responsive relationship between the dose of ketamine and the increase in PROD activity. The levels of hepatic microsomal erythromycin N-demethylation (END), aniline hydroxylation, P450 content, cytochrome b5 and NADPH-P450 reductase were also increased in rats treated with ketamine 80 mg/kg ip twice daily for our days. The increased levels of PROD, ECOD, MROD and END returned to the pre-induction levels four days after the last dose of ketamine administration. On the other hand, the increase in the activities of EROD and aniline hydroxylation remained higher than the control values four days after the last dose of ketamine administration. Protein blot analysis of liver microsomal proteins revealed that 10, 20, 40, 80 mg/kg ketamine induced CYP2B1/2 by 5-, 6-, 10- and 2-fold, respectively. The amount of CYP1A1/2, 2E1 and 3A proteins was increased by 2-fold after treatment of 80 mg/kg ketamine. The polymerase chain reaction analysis showed a 1.7 fold increase in CYP2B mRNA level after ketamine treatment. In regard to ketamine-related drug interactions, the propofol sleeping time was significantly reduced in ketamine-pretreated rats. The reduction could be effectively reversed by a CYP2B inhibitor orphenadrine. The whole-blood propofol concentration after intravenous infusion declined faster in ketamine-pretreated rats. In ex vivo experiments, the ability of hepatic microsomes to metabolize propofol was enhanced in ketamine-pretreated rats. The enhancement in propofol metabolism could be reversed by the addition of orphenadrine. Regarding the bioactivation of toxicants, the liver damage induced by cocaine or carbon tetrachloride (CCl4) was enhanced after ketamine pretreatment. Orphenadrine could effectively reverse the enhancement of cocaine-induced hepatotoxicity by ketamine but not in the case of CCl4. Neither the detection of inflammatory cytokines nor depletion of Kupffer cells by gadolinium chloride could provide a mechanistic explanation for the potentiation of CCl4 toxicity by ketamine. In conclusion, ketamine can induce several P450 proteins; where CYP2B is the most responsive isoform. CYP2B induction is the major mechanism of reduced anesthetic effect of propofol and enhanced propofol metabolism after ketamine treatment in rats. CYP2B induction also plays a principal role in the potentiation of cocaine-induced hepatotoxicity by ketamine.