Biotransformation 생체전환

지난 시간에 Xenobiotics의 특성을 공부하였습니다.

일반적으로 1 kd이하이다. 유기화합물이다. Lipophilic 하다. 비활성 상태로 체내에 들어온다. 체내에서 생체전환된 후 배설되거나 독성을 나타낸다. 체내의 거대분자와 비가역적 상호작용을 통해 독성을 나타낸다.

그리고 독성이 없던 xenobiotics가 체내에서 독성이 생기는 이유도 Xenobiotics 인체 내로 들어오기 위해 얼마나 많은 막을 건너야 하는지도 Xenobiotics가 독성을 나타내기 위해 생체 내 거대분자와 어떤 상호작용을 하는지도 일급발암물질을 약으로 사용하고 있는 이유도 알고 있다.

Principles of toxicology

Principles of toxicology

Biotransformation

Biotransformation

Drug metabolism also known as xenobiotic metabolism is the biochemical modification of pharmaceutical substances or xenobiotics respectively by living organisms, usually through specialized enzymatic systems. Drug metabolism often converts lipophilic chemical compounds into more readily excreted hydrophilic products. The rate of metabolism determines the duration and intensity of a drug's pharmacological action.

Biotransformation

Xenobiotic metabolism (from the Greek xenos "stranger" and biotic "related to living beings") is the set of metabolic pathways that modify the chemical structure of xenobiotics. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects.

Biotransformation

The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment.

Biotransformation

Drug metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.

Highly lipophilic 쉽게 체내에 유입 지방에 축적 생체전환이 잘되지 않는다. 반감기가 길다. Lipophilic 대부분의 Xenobiotics 쉽게 체내에 유입 생체전환이 되어 배출된다

Polar 체내에 유입이 잘 되지 않는다 생체전환 (제2상반응)되어 배출된다 Hydrophilic 체내에 유입이 잘 되지 않는다 유입되어도 바로 배출된다

Polar

외인성물질은 제1상반응에 의해 극성 (polar)을 갖게 된다.

극성은 제2상 반응이 일어나기 위해 필요하다. 극성은 전자를 끌어당기는 힘이다. 전하분포가 불균일할 때 발생한다.

산소와 수소가 결합할 때 산소의 원자핵이 전자를 끌어당겨 부분적으로 음전하를 띠게 되고, 수소는 부분적으로 양전하를 띠게 된다.

Polar

북극곰: 도와줘 나 녹는다! 그냥 곰: 야! 곰은 안 녹아! 북극곰: 너는 그냥 곰이니까 쉽게 말하겠지!

In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure. Polarity underlies a number of physical properties including surface tension, solubility, and melting- and boiling-points.

A water molecule, a commonly used example of polarity. The two charges are present with a negative charge in the middle (red shade), and a positive charge at the ends (blue shade).

This classification table gives a good general understanding of predicting molecular dipole of some general molecular structures. However, one should not interpret it literally:

Predicting molecule polarity

지금까지 Polarity of xenobiotics 를 배웠는데 polarity와 toxicity와의 관계를 설명해 보세요? 일반적으로 xenobiotics는 polar하지 않다. Phase 1 반응에 의해 polar group이 노출되거나 도입된다. Polar group이 있으면 Phase 2에 의해 conjugation되어 배출된다. Polar group이 macromolecule을 공격한다. 결론적으로 hyrophilic하게 만들어 배출하기 위해 polarity를 부여한 결과 독성이 유발될 수 있다.

기억나세요? 지난 시간에…

Lipophilic한 독성물질이 체내로 들어와서는 hydrophilic하게 생체전환되어 배출된다. 결국 체내에 들어온 외인성 물질을 배출하기 위해 생체전환 시킨 결과, 비활성이던 물질이 활성을 가지게 되어 거대분자와 상호작용하여 독성을 유발하게 된다. 배설 (해독) 하려 했는데 독성이 생겼다?

잠깐 쉽시다!

외인성물질을 우선적으로 생체대사시켜 배설하기 위해서는 생체대사 효소가 어디에 많이 있어야 할까요? 왜요? 가장 많은 흡수 경로는 위장관을 통한 것이므로 위장관에 있어야 할 것 같다. (제3의 해독기관?) 위장관의 모든 정맥혈이 간문맥을 통해 일단 간을 거쳐가기 때문에 간에 가장 많을 것 같다. Xenobiotic의 90% 이상 간에서 대사

Biotransformation의 단계 Phase I and II reaction

제1상 반응 Xenobiotics가 산화 (oxidation), 환원

(reduction) , 가수분해 (hydroxylation) 의해 극성 (polar)을 갖게 된다.

-OH, -COOH, -SH, -O- or –NH2 등의 작용기 도입

Cytochrome P450 (P450) 약 80-90% 처리 Flavin-containing monoxygenase (FMO)등

제2상 반응 Xenobiotics의 극성부위에 sulfate,

glucuronide, glutathion등이 포합 (conjugation) 된다.

UDP-glucuronosyltransferase (UGT) 30% Sulfotransferase (SULT) N-acetyltransferase (NAT) Methyltransferase (MT) Gutathione-S-transferase (GST)

제1상 반응에 관여하는 효소 (CYP450)와 제 2상반응에 관여하는 효소의 세포 내 localization 은? Microsome (SER) Cytosol 왜 그럴까요? 지질에 대한 용해성이 높은 외인성물질은 주로 microsome에서 대사된다.

Phase I Cytochrome P450 Microsome Phase II enzyme Cytosol, microsome, mitochondria

Microsome In cell biology, microsomes are vesicle-like artifacts re-formed from pieces of the endoplasmic reticulum (ER) when eukaryotic cells are broken-up in the laboratory; by definition, microsomes are not ordinarily present in living cells.[1]

Microsome Microsomes can be concentrated and separated from other cellular debris by differential centrifugation. Unbroken cells, nuclei, and mitochondria sediment out at 10,000g, whereas soluble enzymes and fragmented ER, which contains cytochrome P450 (CYP), remain in solution (g is the Earth's gravitational acceleration). At 100,000g, achieved by faster centrifuge rotation, ER sediments out of solution as a pellet but the soluble enzymes remain in the supernatant.

S9 fraction The S9 fraction is the product of an organ tissue homogenate used in biological assays. The S9 fraction is most frequently used in assays that measure the metabolism of drugs and other xenobiotics. The "Supernatant fraction obtained from an organ (usually liver) homogenate by centrifuging at 9000 g for 20 minutes in a suitable medium; this fraction contains cytosol and microsomes." The microsomes component of the S9 fraction contain cytochrome P450 isoforms (phase I metabolism) and other enzyme activities. The cytosolic portion contains the major part of the activities of transferases (phase II metabolism).[2] The S9 fraction is easier to prepare than purified microsomes.[3]

Microsome과 S9 fraction을 어디에 적용할까? The S9 fraction has been used in conjunction with the Ames test[4] to assess the mutagenic potential of chemical compounds.[5] Chemical substances sometimes require metabolic activation in order to become mutagenic. Furthermore the metabolic enzymes of bacteria used in the Ames test differ substantially from those in mammals. Therefore to mimic the metabolism of test substance that would occur in mammals, the S9 fraction is often added to the Ames test. The S9 fraction has also been used to assess the metabolic stability of candidate drugs.[6]

Ames test[4]

The Ames test is a biological assay to assess the mutagenic potential of chemical compounds.[1] A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, since cancer is often linked to mutation. The Ames test is often used as one of the initial screens for potential drugs to weed out possible carcinogens, and it is one of the eight tests required under the Pesticide Act (USA) and one of six tests required under the Toxic Substances Control Act (USA).[

Ames test[4]

Rat liver extract is optionally added to simulate the effect of metabolism, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are.[3

Ames test[4] 생각해 볼까요? 장점은 무엇인지? 단점은 없는지?

Ames test[4]

However, a number of false-positives and false-negatives are known.[2] The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound since standard carcinogen assays on rodents are time-consuming (taking two to three years to complete) and expensive. The procedure is described in a series of papers from the early 1970s by Bruce Ames and his group at the University of California, Berkeley.[3][

Ames test[4] Limitations[edit source | editbeta] Salmonella typhimurium is a prokaryote, therefore it is not a perfect model for humans. Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the mutagenic potential of metabolites formed by a parent molecule in the hepatic system can be assessed, however there are differences in metabolism and mutagenicity of chemicals between human and rat.[18] The test may therefore be improved by the use of human liver S9 fraction; its use was previously limited by its availability, but it is now available commercially and therefore may be more feasible.[19] An adapted in vitro model has been made for eukaryotic cells, for example yeast.

Ames test[4] 고용량으로 동물실험해서 저용량 노출에 의한 독성을 평가한다고 했었는데… Bruce Ames himself argued against linear dose-response extrapolation from the high dose used in carcinogenesis tests in animal systems to the lower dose of chemicals normally encountered in human exposure, as the results may be false positives due to mitogenic response caused by the artificially high dose of chemicals used in such tests.

Biotransformation의 결과

Biotransformation의 결과

Biotransformation의 결과 Xenobiotics이 극성, 친수성으로 전환되어

bioinactivation 된다. 친전자성 (electrophilic) 물질로 전환되어 네 가지 거대분자의 친핵성 (nucleophile)부위를 공격한다.

친전자성 대사체는 전자가 부족한 활성중간대사물질을 의미한다.

친전자성 대사체는 GSH conjugation에 의해 친수성으로 전환되어 배출된다.

생체전환에 의해 독성이 일어난다면, 생체전환을 시키지 않으면 되지 않나요? 생체전환을 시키지 않으면 1. 2. 3. 그렇다면 결론은 얻는 게 있으면 잃는 것이 있다?

약의 경우에는 생체전환과 어떤 관계가 있을까요? 약도 독처럼 생체전환 되어야 활성 (약효)이 나올까요? 1.

정리하면 Biotransformation과 bioactivation을 구분할 수 있다. 물질의 특성에 따라 ADME가 달라짐을 알고 있다. 독성과 극성의 관계를 알고 있다. 또한 극성과 대사와의 관계를 알고 있다. Xenobiotics가 biotransformation되는 곳을 알고 있다. Biotransformation의 두 단계를 알고 있다. Phase 1과 phase 2를 구분할 수 있다. Microsome과 S9 fraction의 개념을 알고 있다. Electrophiles의 공격력과 대사 기전을 알고 있다.

밥 먹고 합시다!