Defence

Question 1

types of body defence

Answer 1

Cell permeability

Detoxification

Question 2

Cell permeability

Answer 2

Cell membranes offer a selective absorption barrier

• Water soluble molecules via Active transport or Facilitated diffusion
• Lipid soluble molecules via Passive diffusion

Question 3

Detoxification

Answer 3

Cells need a defence against lipophilic toxicants

Achieved by conversion of lipophilic molecules to hydrophilic metabolites

Toxicant –> phase I –> phase II –> urinary excretion / Faecal excretion

Question 4

Phase I reactions

Answer 4

Generally introduce a reactive oxygen or nitrogen into a toxicant – becomes a substrate for Phase II metabolism

Catalysed by oxidative, reductive or hydrolytic enzymes, generally in smooth ER in lungs, liver and intestines

Reactions reduce lipophilicity and render toxicants nucleophilic

Cellular macromolecules (DNA, RNA and proteins) are also nucleophilic – renders toxin less reactive

Can often predict likely metabolites – can be part of safety assessments

Question 5

Phase I Reductive reactions

Answer 5

Carbonyl reductase acts on Carbonyl residue (=O) to - OH

```
Cytochromes P450 (CYP) + low O2 ::
-NO2) –> (-NH2
(N=N) –> 2x (-NH2)
~~~

Question 6

Phase I Oxidative reactions-

Answer 6

Cytochromes P450 (CYP) added on COH for phenyl and alkane

• for alkene, Cytochromes P450 (CYP) added on O to half filled the double bond, then Epoxide hydrolases act on the epoxide residue and go through a dihydroxylation reaction to form diol products

Question 7

Phase I Dealkylation reactions -

Answer 7

cleave off CH2 by Cytochromes P450 (CYP)

Question 8

Phase I Enzymes

Answer 8

Cytochrome P450 (CYP) (+/without low O2)
Epoxide hydrolases
Carbonyl reductase
Flavin-Containing Monooxygenase

Question 9

Cytochrome P450 (CYP)

Answer 9

Haeme containing enzymes absorb at 450nm

> 400 enzymes in 36 families

Richest source is the liver

Human genome sequence has 56 CYP – mostly involved in metabolism of endogenous substances

Found in all species

CYP is part of a membrane-bound complex with CYP reductase

Question 10

CYP mechanism

Answer 10

slide 23

Question 11

Ex of CYP enzyme

Answer 11

CYP3A4 / CYP1B1 / CYP2E1

Question 12

CYP3A4 location (2) & substrate (4)

Answer 12

Liver & Intestine

Flavonoids / Aflatoxins / Pesticides / Food additives

Question 13

CYP1B1 location & substrate

Answer 13

Wide spread in many tissue

Polycyclic aromatic hydrocarbons /
Arylamines / Procarcinogens

Question 14

CYP2E1 location & substrate

Answer 14

Liver
Inducible in many tissues by ethanol

Ethanol, acetone and acetaldehyde
Acrylamide

Question 15

CYP enzymes can activate _________

Inhibited by ________

Answer 15

CYP enzymes activate several important carinogens

Inhibited by phytochemicals:

• Resveratrol (grapes)
• Genestein and equol (soy)
• Diallyldisulfides (garlic)

Question 16

Phase I : Flavin-Containing Monooxygenase

Answer 16

FMO selectively oxidise nucleophilic heteroatoms in lipophilic toxicants

Produce more polar metabolites – more readily excreted
Expressed at high level in liver, kidney and lung

Nicotine –> Nicotine-1’-N-oxide

Question 17

Phase II Metabolism

Answer 17

Converts functionalised metabolites from Phase I into excretable products

Increase water solubility

Decrease toxicity

Generally involves addition of a polar endogenous molecule

Question 18

polar endogenous molecule required in Phase II Metabolism (3)
& reaction involved

Answer 18

Glucuronic acid –> Glucuronidation
Sulfate –> sulfation conjugate nucleophiles
Glutathione –> glutathione conjugates electrophiles

Question 19

Glucuronide conjugation

Answer 19

Major pathway conjugating alcohols, phenols, carboxylic acids, amines and thiols

Enzyme is uridine diphosphate-glucuronyl transferase (UGT)
works with Uridine diphosphoglucuronide (UDP-GA)

• -> to cleave (-COOH)/ (-OH)/ (NH2) / (SH) off (-R)
• -> transfer -R onto a sugar ring
• -> Active transport out of liver by the organic anion transporter (OAT)
• -> if <350 Da , excrete via urine / if >350 Da , excrete via faecal

Question 20

(UGT) is found in

Answer 20

uridine diphosphate-glucuronyl transferase (UGT)

Found in smooth ER membrane in liver, intestine, brain, skin, spleen and nasal mucosa

Question 21

Sulphate conjugation

Answer 21

removes alcohols, phenols, amines, hydroxylamines but not carboxylic acids

Sulfotransferase enzyme uses a sulfate donor
and
works with 3’-Phosphoadenosine-5’-phosphosulfate (PAPS)

• -> removes (-OH) / (-NHOH) / (-NH2) and link (-R) with sulfate
• -> Active transport via kidneys by the organic anion transporter (OAT)
• -> Urinary excretion

Question 22

Compare Sulfotransferase to uridine diphosphate-glucuronyl transferase

Answer 22

Sulfotransferase (ST) is a high affinity, low capacity enzyme – removes lower concentrations of toxicants than UGT

both removes alcohols, phenols, amines
ST also removes hydroxylamines
UGT also removes carboxylic acids and thiols

Question 23

Glutathione conjugation

Answer 23

Glutathione conjugates to a wide range of electrophilic toxicants

Enzyme is glutathione-S-transferase (GST)
works with Glutathione (GSH)

Conjugates can be converted to a mercapturic acid derivative in the kidney – increases efficiency of excretion

e. g. epoxide toxicant had coverted into an alkane (with - GS/ - OH instead) through phase I
- -> GST + GSH convert the conjugate into a mercapturic acid and excrete via urine
- -> rather than Active transport into bile by the organic anion transporter (OAT) and excrete via faeces