Biotransformation

생체전환
지난 시간에 Xenobiotics의 특성을 공
부하였습니다.
일반적으로
1 kd이하이다.
유기화합물이다.
Lipophilic 하다.
비활성 상태로 체내에 들어온다.
체내에서 생체전환된 후 배설되거나 독성을 나
타낸다.
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 거대분
자와 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 전자
를 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).
Predicting molecule polarity

This classification table gives a good general

understanding of predicting molecular dipole of
some general molecular structures. However, one
should not interpret it literally:
지금까지 Polarity of xenobiotics 를 배웠
는데 polarity와 toxicity와의 관계를 설명
해 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 위
해 polarity를 부여한 결과 독성이 유발될 수 있
다.
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 배
설하기 위해서는 생체대사 효소가 어디에
많이 있어야 할까요? 왜요?

가장 많은 흡수 경로는 위장관을 통한 것이
므로 위장관에 있어야 할 것 같다. (제3의
해독기관?)

위장관의 모든 정맥혈이 간문맥을 통해 일

단 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 주

로 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 평
가한다고 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 가
지 거대분자의 친핵성 (nucleophile)부위를
공격한다.

친전자성 대사체는 전자가 부족한 활성중간대

사물질을 의미한다.

친전자성 대사체는 GSH conjugation에 의해 친

수성으로 전환되어 배출된다.
생체전환에 의해 독성이 일어난다면, 생
체전환을 시키지 않으면 되지 않나요?

생체전환을 시키지 않으면

1.
2.
3.

그렇다면 결론은 얻는 게 있으면 잃는 것

이 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!