BIOCHEMISTRY

LECTURE # 3.07: Metabolism of Xenobiotics

DATE OF LECTURE: February 22, 2018
REPORTERS: Rodrigo, Punzalan, Ticman | INSTRUCTOR: Dr. Marzan

OUTLINE • One atom of oxygen of molecular O2 contributes to the hydroxyl

group of R-OH, and the other forms water → dual fate of oxygen
I. Xenobiotics • Accounts for the name mixed-function oxidase
II. Metabolism of Xenobiotics
A. Phase I CYTOCHROME P450 CATALYTIC ACTION
B. Phase II 1. Oxidized (Fe3+) P450 combines with a drug substrate to form a
III. Factors Affecting Metabolism of Xenobiotics binary complex.
IV. Responses of the Body to Xenobiotics 2. NADPH donates an electron to the flavoprotein P450 reductase,
V. Pharmacogenomics which in turn reduces the oxidized P450-drug complex.
3. A second electron is introduced from NADPH via the same P450
reductase, which serves to reduce molecular oxygen and to form an
I. XENOBIOTICS “activated oxygen”-P450-substrate complex.
• Xenobiotics are compounds foreign to the body. 4. This complex in turn transfers activated oxygen to the drug
• Primary classes of xenobiotics that are of medical significance are substrate to form the oxidized product.
drugs (eg R Thalidomide), chemical carcinogens (eg S
Thalidomide), naturally-occurring compounds in plant foods (eg
Beta-carotene), and several other compounds (eg Tear gases),
such as Polychlorinated biphenyls (PCBs), insecticides, and other
pesticides (ie Agent Orange).

II. METABOLISM OF XENOBIOTICS

• The ultimate goal of metabolizing xenobiotics: convert it in
inactive, polar (water soluble), non-toxic formmore readily
excreted in water or bile
o Exceptions:
▪ Pro-drugs: initially inactive; converted to their active
forms once metabolized
▪ Formation of toxic metabolites: Ex. N-acetyl-p- Figure 1. Catalytic action of cytochrome P450.
benzoquinone imine (NAPQI) is a hepatotoxic byproduct
produced during the metabolism of the Paracetamol HYDROXYLATION
• Main organ of detoxification: Liver • Requirements:
• Two phases of metabolism: o P450
o Phase I (Goal: unmask or insert polar functional group) o P450 Reductase
o Phase II (goal: add a polar conjugate) o NADPH
o Molecular Oxygen
• Reaction:
A. PHASE I o RH + O2 + NADPH + H+ → ROH + H2O + NADP
• Major reaction involved: • Substrate RH:
o Hydroxylation (lumabas sa quiz!) o Generally lipophilic
• Other reactions involved: o Made more hydrophilic through the Phase 1 reaction
o Deamination o May represent both xenobiotic (drugs, pesticides), or
o Dehalogenation endogenous compounds (eicosanoids, fatty acids)
o Desulfuration
o Epoxidation B. PHASE II
o Peroxygenation
o Reduction • Phase 1 reactions usually, but not always, precedes Phase 2
o Hydrolysis reactions.
• Catalyzed mainly by a class of enzymes: Monooxygenases or • Renders the products from Phase 1 to be even more water soluble,
cytochromes P450 and they are eventually excreted in the urine or bile.
• Types:
CYTOCHROME P450 o Glucuronidation
• Most versatile biocatalyst known o Sulfation
• Responsible for the metabolism of approximately 50% of the
o Conjugation with glutathione
common drugs ingested by humans
o Other reactions (Acetylation, Methylation)
• Binds with carbon monoxide in its reduced form
• Complex exhibits an absorption peak at 450 nm

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 BIOCHEMISTRY
LECTURE # 3.07: Metabolism of Xenobiotics
DATE OF LECTURE: February 22, 2018
REPORTERS: Rodrigo, Punzalan, Ticman | INSTRUCTOR: Dr. Marzan

1. GLUCURONIDATION • Function:
• Most frequent conjugation reaction o Cellular defense
• Same way as glucuronidation of bilirubin o Reduction of H2O2 to water
• UDP-glucuronic acid is the glucuronyl donor and other variety of o Maintains thiol (-SH) group in reduced state
glucuronosyltransferases present in the ER and cytosol. o Metabolism of xenobiotic compounds
o Carrier of certain amino acids across membrane in kidney
• Excreted as glucuronides, may be attached to oxygen, nitrogen, or
sulfur groups of the substrates
• Xenobiotics excreted as glucuronide:
o 2-acetylaminofluorene (carcinogen)
o Aniline
o Benzoic acid
o Meprobamate (tranquilizer)
o Phenol
o Many steroids

Figure 4. Conjugation of Glutathione.

• Toxic electrophilic xenobiotics are conjugated to the nucleophilic

GSH in reaction:
Figure 2. Glucuronidation of aniline to aniline n-glucuronide. o R + GSH → R–S–G
o R: electrophilic compound
2. SULFATION o Enzyme: glutathione S-transferases
o GSH: because of the sulfhydryl group of cysteine
• Some alcohols, arylamines, phenols are sulfated.
• When not conjugated with GSH, xenobiotics are free to covalently
• Sulfate donor:
combine with DNA, RNA, or cell protein thus causing serious cell
o Adenosine 3’-phosphate-5’-phosphosulfate (PAPS)
damage
o “active sulphate”
• Before excretion, glutamyl and glycinyl parts are removed and an
• Present in these and other biologic and sulfation reactions (e.g.
acetyl group (from acetyl-CoA) is added to the amino group of the
sulfation of steroids, glycosaminoglycans, glycolipids, and
cysteinyl moiety.
glycoproteins)
• Resulting compound: Mercapturic acid
• Sulfated substrates:
o Alcohol
4. ACETYLATION
o Arylamines
o Phenols • Acetyl-CoA is the acetyl donor
o Steroids • Catalyzed by Acetyl Transferases
o Glycosaminoglycans (GAGS) • Present in the cytosol of several tissues (particularly in the liver)
o Glycolipids • With polymorphic types of acetyltransferases
o Proteins • Influence the rate of clearance of drugs (e.g. isoniazid)
• This reaction is represented by:
o X + Acetyl-CoA → Acetyl-X + CoA

Figure 3. Tyrosine sulfation reaction (found in bovine). The enzyme

Tyrosylprotein sulfotransferase(TPST) in the trans-golgi compartment
transfers a sulfate group from PAPS.

3. CONJUGATION
• Glutathione (γ-glutamyl-cysteinylglycine) is a tripeptide consisting
of Glutamic acid, Cysteine, and Glycine (Mnemonic: ECG) Figure 5. Acetylation of Isoniazid drug.
• Forms glutathione S-conjugates that are excreted in the urine and
bile

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 BIOCHEMISTRY
LECTURE # 3.07: Metabolism of Xenobiotics
DATE OF LECTURE: February 22, 2018
REPORTERS: Rodrigo, Punzalan, Ticman | INSTRUCTOR: Dr. Marzan
5. METHYLATION
• Methyl donor is S-Adenosylmethionine (SAM)
• Catalyzed by methyltransferases
• Compounds conjugated by methylation:
o Nicotinamide
o P-methylaminoazobenzene
o Dimethylaminoazobenzene
o Estrogen
o Norepinephrine
o Epinephrine and their metabolites
III. FACTORS AFFECTING METABOLISM OF XENOBIOTICS
• Variety of factors affect the activity of the enzymes involved in
metabolizing xenobiotics
• As such, activities of the metabolizing enzymes of xenobiotics differ
substantially between species therefore toxicity and carcinogenicity
cannot be concluded in a universal way
A. GENETIC FACTORS
• Main factor in metabolism of xenobiotics
• Mutations that affect transcription which lead to disorders/diseases
• As in mutation in genes of cytochrome P450 resulting to over-
metabolism and under-metabolism of the xenobiotic agent
• Result in drug toxicity, tissue injury, variable drug effects and
various drug-drug interactions
Figure 6. Genetic Factors affecting Xenobiotic metabolism
B. AGE
• As an organism age, activity of enzymes as well as other
bodily functions deteriorate.
• Intake of some xenobiotics may also cause enzyme induction.
Cadmium, as an example, is absorbed greater in newborn rats than
in adults, and is retained in the intestine for a longer time (Sasser,
1977).
• Development and activity of enzymes such as N demethylase,
benzpyrene hydroxylase, biphenyl 4-hydroxylase,
ethoxycoumarin O-deethylase, NADPH-cytochrome c reductase
activities, and cytochrome P-450 content are also age-dependent.
C. SEX
• Males and females release different amounts and kinds of
hormones so this affects the metabolism of xenobiotics as
well.
Trans By: Rodrigo, Punzalan, Ticman
• In general, when a sex-dependent difference is observed in
humans, females have higher plasma concentrations of the drug as
compared with men.
D. DISEASE
• Various diseases affect the activities of drug-metabolizing
enzymes
• In liver cirrhosis, activities of drug-metabolizing enzymes are
affected
E. INTAKE OF OTHER XENOBIOTICS
• Most important in practice (esp. drug-drug interaction)
• Enzyme Induction and Inhibition
o Enzyme Induction: stimulates enzymatic
activitymetabolism of xenobiotics (drugs)
▪ Consequence:  activity of drug
efficacysubtherapeutic effect
▪ Ex. Rifampicin (anti-TB drug) induces the metabolism of
antiviral drugs taken by HIV patients. (Alternative:
Rifabutin)
o Enzyme Inhibition: decreases/inhibits enzymatic
activitymetabolism of xenobiotics (drugs)
▪ Consequence:  plasma concentrationToxicity
IV. RESPONSES OF THE BODY TO XENOBIOTICS
CYTOTOXICITY
● Ability to destroy living cells which can be severe enough to result
in cell death
● Covalent binding to cell macromolecules (DNA, RNA and protein)
of reactive species of xenobiotics produced by metabolism
o Example: Cyclophosphamide
▪ Alkylating agent used in cancer chemotherapy
▪ Upon its metabolism, its byproducts (alkyl radicals) are
incorporated into DNA strands resulting to an interstrand
DNA adductsinhibition of DNA replicationcell death
ACT AS HAPTEN
● Reactive metabolite of a xenobiotic may bind to a protein, acting
as a hapten, and altering its antigenicity, causing a potentially
imitating autoimmune disease or allergy
o Example: β Lactams (Penicillins)
▪ reacts chemically with proteins in the body to form a
hapten-carrier complexallergic reactions (anaphylaxis)
CHEMICAL CARCINOGENS
7-
● Activities of the xenobiotic metabolizing enzymes present in the
endoplasmic reticulum thus help to determine whether such
compounds become carcinogenic (cancer) or are “detoxified”
o Example: benzo[α]pyrene
▪ highly carcinogenic polycyclic aromatic hydrocarbon
(PAH)
▪ requires activation by monooxygenases in the
endoplasmic reticulum to become carcinogenic (indirect
carcinogens)
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 BIOCHEMISTRY
LECTURE # 3.07: Metabolism of Xenobiotics
DATE OF LECTURE: February 22, 2018
REPORTERS: Rodrigo, Punzalan, Ticman | INSTRUCTOR: Dr. Marzan

V. PHARMACOGENOMICS C. DEVELOPMENT OF NEW DRUG TARGETS

● Study of the contribution of genetic factors to variation in drug ● Major thrusts of new drug development: to enhance treatment and
response and toxicity to provide safer, personalized drugs, taking into account
● Used interchangeably with pharmacogenetics polymorphisms and other genetic and environmental factors.
● It builds on pharmacogenetics, but covers a wider sphere of activity ● Detection of potentially harmful polymorphism can be done through
microarrays permitting screening of individuals prior to the start of
Table 1. Pharmacogenomics vs Pharmacogenetics drug therapy.
PHARMACOGENOMICS PHARMACOGENETICS o Gene chips are microscope slides printed with thousands of
tiny spots in defined positions, with each spot containing a
Definition Systematic identification of Science of determining known DNA sequence or gene.
all the human genes, the genetic differences o These are available for analyzing certain P450 genotypes.
their products, inter- on metabolic pathways
individual and intra- which can affect REFERENCES
individual variation in individual responses
expression and function to drugs • Alexandrov, K., Rojas, M. and Rolando, C. (2006). DNA Damage by
Goal Increase efficiency of Tailor medical Benzo(a)pyrene in Human Cells Is Increased by Cigarette
drug development treatments to the Smoke and Decreased by a Filter Containing Rosemary Extract,
process individual Which Lowers Free Radicals. Cancer Research, [online] 66(24),
pp.11938-11945. Available at:
http://cancerres.aacrjournals.org/content/66/24/11938 [Accessed
A. SINGLE NUCLEOTIDE POLYMORPHISM (SNPS) 21 Feb. 2018].
● Variation in a genetic sequence that affects only one of the basic • Boelsterli, U. (2009). Mechanistic toxicology. 2nd ed. New York:
building blocks (nucleotides) Informa Healthcare.
● It is the most common type of genetic variation among people • Encyclopedia Britannica. (2018). Single nucleotide polymorphism |
● occur once in every 300 nucleotides ( on average) genetics. [online] Available at:
● When SNPs occur within a gene or in a regulatory region near a https://www.britannica.com/science/single-nucleotide-polymorphism
gene, they may play a more direct role in disease by affecting the [Accessed 21 Feb. 2018].
gene’s function • Patil, J. (2015). Pharmacogenetics and Pharmacogenomics: A Brief
● Applications: Introduction. Journal of Pharmacovigilance, [online] 03(03).
o Track susceptibilities to common diseases (cancer, HTN, Available at: https://www.omicsonline.org/open-
diabetes) access/pharmacogenetics-and-pharmacogenomics-a-brief-
o Identify new drug targets and diagnostic tests introduction-2329-6887-1000e139.php?aid=54556 [Accessed 21
o As biomarker for drug response Feb. 2018].
• Rodwell, V., Bender, D., Botham, K., Weil, P., Kennelly, P. and
B. APPLICATION OF PHARMACOGENOMICS Murray, R. (2009). Harper's Illustrated Biochemistry. 28th ed.
McGraw-Hill Publishing.
• Rost, S., Fregin, A., Ivaskevicius, V., Conzelmann, E., Hörtnagel, K.,
DRUG TARGETING: WARFARIN Pelz, H., Lappegard, K., Seifried, E., Scharrer, I., Tuddenham, E.,
● Warfarin targets the vitamin K epoxide reductase multiprotein Müller, C., Strom, T. and Oldenburg, J. (2004). Mutations in
complex subunit 1 (VKORC1) VKORC1 cause warfarin resistance and multiple coagulation factor
● VKORC1 recycles vitamin K 2,3-epoxide to vitamin K hydroquinone deficiency type 2. Nature, 427(6974), pp.537-541.
o Vit K: a cofactor needed to activate coagulation factors X, IX, • Walker, K., Ginsberg, G., Hattis, D., Johns, D., Guyton, K. and
VII, II. Sonawane, B. (2009). Genetic Polymorphism inN-Acetyltransferase
● Patients with VKORC1 missense mutations are warfarin (NAT): Population Distribution of NAT1 and NAT2 Activity. Journal
“resistant” and require higher doses of warfarin. of Toxicology and Environmental Health, Part B, [online] 12(5-6),
pp.440-472. Available at:
DRUG METABOLISM: ISONIAZID https://www.ncbi.nlm.nih.gov/pubmed/20183529 [Accessed 21 Feb.
● Metabolized by N-Acetyltransferases (NAT) 2 2018].
● Multiple NAT2 alleles (NAT2*5, *6, *7, and *14): substantially
decreased acetylation activity
o Common in Caucasians and populations of African descent Uy. Pagod ka na? Pahinga ka muna. Tapos aral ulit, ha?
o Slow acetylators: more susceptible to the side effects
mediated by acetylated metabolites Don’t be too hard on yourself. Stay DETERMINED,
● NAT2*4 allele: increased acetylation
o Common in majority of Eskimos and Asians as a famous game always say (Laruin niyo, Undertale).
o Fast acetylators: may not reach therapeutic levels with
treatment and require greater doses -J
o Leads to low drug plasma concentration of isoniazid

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 BIOCHEMISTRY
LECTURE # 3.07: Metabolism of Xenobiotics
DATE OF LECTURE: February 22, 2018
REPORTERS: Rodrigo, Punzalan, Ticman | INSTRUCTOR: Dr. Marzan

A. Increases B. Decreases
LONG QUIZ
1. The following involves use of Xenobiotics, EXCEPT 18. Who has higher rates of metabolism of xenobiotics? (2 choices)
A. Chlorine gas used in the Syrian Gas Attack A. Males B. Females
B. Agent Orange sprayed on crops during Vietnam War
C. Taking up Fluoxetine 19. Therefore, answer in 19, in general, must be given ______ doses? (2
D. IV injection of Piperacillin/Tazobactam (Piptaz) Choices)
E. All choices involves Xenobiotics A. Higher B. Lower

2. The following are involved in Cytochrome P450, EXCEPT 20. The following is true of Pharmacogenomics, EXCEPT
A. NADPH B. NADH C. Fe3+ D. Fe2+ E. O2 A. Deals with Genomics, which deals with all genes
B. Aim to increase efficiency of drug development process
3. The following belong to the Phase I reactions, EXCEPT C. Tailor medical treatments to the individual
A. Hydroxylation B. Deamination C. Reduction D. Acetylation D. None of the above

4. The following belong to the Phase II reactions, EXCEPT For nos 21-25
A. Glucoronidation B. Acetylation A. Warfarin B. Isoniazid C. Paracetamol
C. Dehalogenation D. Methylation
21. Targets the vitamin K epoxide reductase multiprotein complex subunit 1
5. End product of Conjugation? (VKORC1)
A. Mercaptoethanol B. Mercapturic acid 22. Byproduct is N-acetyl-p-benzoquinone imine
C. N-acetyl-p-benzoquinone imine D. Paracetamol 23. Requires higher doses in Caucasians and those with African descents

6. The following are excreted as glucoronides, EXCEPT MGA SAGOT SA TANONG KUNG BAKIT KA MAHALAGA
A. 2-Acetylaminofluorene (carcinogen) B. Aniline 1. A. Xenobiotics are Compounds, not elements. Chlorine gas, Cl2, is an
C. Benzoic acid D. Alcohol elemental gas, not a compound. Never forget your Chemistry.
2. B. The rest are involved in Cytochrome P450
7.The following are requirements in Phase I reaction, EXCEPT 3. D. Acetylation belongs to the Phase II reactions. Sa Phase I, you are
A. P450 B. P450 Reductase C. O2 D. FADH2 simply adding or removing functional groups. Sa Phase II, nagkakabit ka
ng pampa-polar (eg Acetyl group)
8. The following is true of 7, EXCEPT 4. C. Dehalogenation belongs to the Phase I reactions. AGAIN, Phase I is
A. The goal is to make the xenobiotic more water-soluble centered on functional groups. Phase II is centered on adding a polar
B. In a clinical case where patient has decreased PO2, Phase I reaction compound or a molecule. Gets? Okay? Okay.
efficiency is decreased 5. B. If you answered D, gisingin mo muna sarili mo. A is a different
C. The goal is to make the xenobiotic more lipid-soluble compound. C is the product of the metabolism of D
D. In an overdrive of Phase I reaction, higher doses of the drug must be 6. D. Okay, ganito yan, yung alcohols, they are essentially nucleophilic, and
given to compensate for a decrease in Plasma concentration can be used to attack S of Sulfates, hence, are excreted by sulfation. The
rest, conjugated to glucoronide
For nos 9-13 7. D. FADH2 is not even involved in these redox reactions. NADPH is the
A. Glucoronidation B. Sulfation C. Conjugation reducing equivalent used in hydroxylation reactions
D. Acetylation E. Methylation 8. C. Phase I reaction is supposed to make the Xenobiotic more water-
soluble
9. Cellular defense 9. C. Cells make use of conjugation to defend it from harmful substances
10. Donor is S-Adenosylmethionine (SAM) 10. E. Recall Second shifting reactions. SAM is commonly used as a methyl
11. Rate is increased by Glycolysis donor.
12. Epinephrine processing 11. D. You produce Acetyl CoA in Glycolysis, which is used as the Acetyl
13.Involves Glutathione donor in Acetylation reactions. Does it make sense now?
12. E. Epinephrine and Norepinephrines are methylated.
14. The following comprises no. 13, EXCEPT 13. C. Glutathione is involved in conjugation reactions
A. Glutamic Acid B. Cysteine C. Glutamine D. Glycine 14. C. RECALL Mnemonic for Glutathione= ECG
15. A. Genetic factors is the main factor in Xenobiotics metabolism
15. Main factor in metabolism of Xenobiotics 16. C. Recall, you have an overdrive of Cyp450, you break drugs down
A. Genetic Factors B. Age C. Sex faster, hence, you need to increase the dose, not lower it
D. Disease E. Intake of other Xenobiotics 17. B. Napapagod ang enzymes natin, tulad natin. It is human nature to get
tired, take shortcuts, lie.
16. Mutations in the Cytochrome P450 gene lead to overmetabolism of 18. A. Mas mabilis ang metabolism ng males, so they have lower plasma
xenobiotics. This implies the following, EXCEPT? concentrations of the drugs, Therefore…
A. Px (Patient) must not be O2 desaturated 19. A. Higher doses must be given.
B. Higher doses of medication of Px must be given 20. C. When it comes to the individual, Pharmacogenetics is concerned.
C. Lower doses of medications of Px must be given 21. A. This is the target of Warfarin(anticoagulant)
D. Iron (Fe) supplements may be needed 22. C. This is the problem with Paracetamol
23. B. Isoniazid acetylation with these people is substantially decreased
17. As an individual ages, enzyme efficiency (two choices only)

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