Biochemistry Of Pharmtox Test 2

Front 1

What is the function of metabolism?

Back 1

-elimination and detoxification of xenobiotics
-usually increase polarity

Front 2

What are the classifications of metabolic reactions?

Back 2

-phase I reactions-attach a small polar functional group (oxidations, reductions, and hydrolyses
-phase II reactions- attach a small polar molecule (glucuronidation, sulfation, glutathione conjugation, etc)

Front 3

What are metabolic sites for drug metabolism?

Back 3

-every tissue has some capability of drug metabolism
-most occurs in liver
-CYP's are in all cells of the body exceot red blood cels

Front 4

What cells metabolize in the liver? kidney? lung? intestine? skin adn nose?

Back 4

-hepatocytes (zonal distrbution)
-proximal tubular cells
-clara cells, type II cells
-mucosal cells and gut bacteria
-epithelial cells

Front 5

What are the subcellular sites for drug metabolism?

Back 5

-membrane-bound microsomes and mitochondria (endoplasmic reticulum, most pahse I enzymes located here)
-soluble- cytosol (most pahse II enzymes located here)

Front 6

What are "expresed" enzymes? Preparation? advantages? disadvantages?

Back 6

-enzyme is expressed in a cell line
-gene inserted into DNA of host cell
-grow cells and harvest
-isolate subcellular fractions
-can evaluate single isozyme activity, allows use of human enzymes, commercially available
-non-physiological, DNA sequence of gene must be known, technical challenges

Front 7

Whata re isolated cells/.cultured cells? preparation? choice of technique? advantages? disadvanatages?

Back 7

-good in vitro model
-measure metabolisma nd toxicity simultaneously
-organ is enzymatiically digested
-intact cells are collected and used immedatly or cultured
-freshly isolated cells or cultured cells (can differentiate and lose CYP enzymes)
-all metabolic pathways present, simultaneously measure metabolism and cytotoxicity, mutliple incubations from single animal, commercially available
-non-physiological, short lifespan (isolated cells), variable loss of enzyme acitivty (cultured cells)

Front 8

What are subcellular fractions? preparation? advantages? disadvantages?

Back 8

-measure metabolism in microsomes, cytosol, S-9 fraction or mitochondria
-prepare via differential centrifugation of organ homogenates
-separate ER from cytoplasm, can use any tissue in body
-easy preparation, simple meatbolic profile, multiple incubations froma single animal, commercially available
-non-physiological model, possible loss of activity with storage

Front 9

Whata re purified enzymes? advanatges? disadvantages?

Back 9

-purify from subcellular fractions, you cna find which CYP
-evaluate single isozyme activity, comemrcially available
-non-physiological, difficult purification

Front 10

What are animal studies? advantages? disadvanatages?

Back 10

-in vivo
-measure metabolites in tissues, plasma, urine, bile, feces, etc.
-all metabolic pathwasy are present, can measure pharmacokinetics (ADME)
-may require use of radiolabeled compound, expensive, biological variability, etc.

Front 11

What is cytochrome p-450?

Back 11

-enzyme found in virtually all eukaryotic and prokaryotic organisms (no subcellular organelles, cytoplasmic CYP's)
-several thousands of isozymes, mechanisms of action are same
-present in many tissues, found in all cells of body except RBC's
-microsomal, heme-containing enzyme (b/c CYP's are O2 binding, Hb in blood is not enzymatic

Front 12

What is the general reaction of CYP 450? What does it require?

Back 12

-commonly catalyses monooxygenations
-inserts single oxygen atom into substrate
-oxygen source- O2
-cofactor- B-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)
-coenzyme-NADPH cytochrome p-450 reductase
-other- membrane lipids (help to keep the 2 enzymes close to each other

Front 13

What are other reactions CYP 450 do?

Back 13

-desaturations, reductions, etc.

Front 14

Where is CYP isozyme 1A1/1A2 located? Typical substrates?

Back 14

-many (1A1 low in liver
-polycyclic aromatic hydrocarbons (PAH)
-in cigarette smoke

Front 15

Where is CYP isozyme 1B1 located? Typical substrates?

Back 15

-many
-polycyclic aromatic hydrocarbons

Front 16

Where is CYP isozyme 2A6 located? typical siubstrates?

Back 16

-liver
-steroids

Front 17

Where is CYP isozyme 2B6 located? typical substrates?

Back 17

-liver, heart
-nicotine

Front 18

Where is CYP isozyme 2C9/2C10 located? typical substrates?

Back 18

-liver
-tolbutamide

Front 19

Where is CYP isozyme 2C19 located? typical substrates?

Back 19

-liver, heart
-omeprazole

Front 20

Where is CYP isozyme 2D6 located? typical substrates?

Back 20

-liver, heart, brain
-antidepressants and Beta-blockers

Front 21

Where is CYP isozyme 2E1 located? Typical substrates?

Back 21

-many
-acetaminophen (toxicity)

Front 22

Where is CYP isozyme 2F located? typical substrates?

Back 22

-lung
-coumarins

Front 23

Where is CYP isozyme 3A4/3A5 located? typical substrates?

Back 23

-many (adult)
-many

Front 24

Where is CYP isozyme 3A7 located? typicla substrates?

Back 24

-liver (fetal)
-many similar to 3A4

Front 25

Where is CYP isozyme 4A9/4A11 located? typical substrates?

Back 25

-kidney
-fatty acids

Front 26

What percentage of CYP isozyme 1A2 exists? role in metabolism? inducers? inhibitors?

Back 26

-13%
-<4%
-PAH's
-furafylline

Front 27

What percentage of CYP isozyme 2A6 exists? role in metabolism? inducers? inhibitors?

Back 27

-4%
-<1%
-phenobarbital
-no inhibitors

Front 28

What percentage of CYP isozyme 2B6 exists? role in metabolism? inducers? inhibitors?

Back 28

-1%
-<1%
-phenobarbital
-no inhibitors

Front 29

What percentage of CYP isozyme 2C8, 2C9/2C10, and 2C19 exist? role in metabolism? inducers? inhibitors?

Back 29

-20%
-11%
-phenobarbital
-tolbutamide (2C8), quercetin (2C8), sulfaphenazole (2C9/10), mephenytoin (2C19), and omeprazole (2C19)

Front 30

What percentage of CYP isozyme 2D6 exists? role in metabolism? inducers? inhibitors?

Back 30

-4%
-30%
-no inducers
-no inhibitors

Front 31

What percentage of CYP isozyme 2E1 exists? role in metabolism? inducers? inhibitors?

Back 31

-6%
-2%
-ethanol, isoniazid, and starvation
-diethldithiocarbamate

Front 32

What percentage of CYP isozyme 3A4 exists? role in metabolism? inducers? inhibitors?

Back 32

-30%
-52%
-rifampin
-troleandomycin and ketoconazole

Front 33

What oxidative reactions are catalysed by cytochrome P-450?

Back 33

-aliphatic hydroxylation
-aromatic hydroxylation
-epoxidation
-dealkylation
-oxidation on heteroatoms

Front 34

What is aliphatic hydroxylation by cytochrome P450?

Back 34

-occurs at C-H bonds in aliphatic chains (very stonrg bond)

Front 35

What experiment is a good example of aliphatic hydroxylation by cytochrome P450 and what was significant about it?

Back 35

-hydroxylation of valproic acid by rat liver microsomes
-animals treated with phenobarbital before incubated with microsomes
-had NADPH generating system (constanty generates NADPH so rxn isn;t limited)
-take out NADPH to see if metabolism still occurs, if it does, then it;s not CYP mediated
-carbon monoxide inhibited CYP even with phenobarbital induction

Front 36

What is aromatic hydroxylation by cytochrome P450?

Back 36

-occurs at C-H bond in aromatic ring

Front 37

What experiment is a good example of aromatic hydroxylation by cytochrome P450 and what was significant about it?

Back 37

-hydroxylation of warfarin by purified rat liver CYP isozymes
-incubation contained warfarin, CYP, CYP reductase, NADPH, and phospholipids
-isozyme 2C11 is the predominant isozyme in mlae rat liver, not found in female rat liver
-2C6 is induced by phenobarbital, also 1A1
-isozymes discriminate b/t metabolites produced, catalytic cylces are all identical though, probably depends on how warfarin orients itself on the active site of the enzyme

Front 38

What is epoxidation by cytochrome P450?

Back 38

-formation of epoxide ring at carbon-carbon double bonds and ona romatic rings
-monooxygenation

Front 39

What experiment is a good example of epoxidation by cytochrome p450 and what was significant about it?

Back 39

-epoxidation of acrylonitrile by isolated rat lung cells
-potentially carcinogenic to humans, can also be aersolized
-known carcinogen in rats
-cytochrome p450 has predminant role in making epoxide in specific cells, like the lungs

Front 40

What is dealkylation by cytochrome p450?

Back 40

-loss of an alkyl group
-monooxygenation, but doesn't look like one
-oxygen goes on alkyl group cleaved off and becomes an aldehyde
-necer occurs where carbon is surrounded by alkylic groups

Front 41

What experiment is a good example of dealkylation by cytochrome p450? How is it significant?

Back 41

-N-dealkylation of cisapride to nor cisapride
-CYP3A4 are having biggest effect therefore assume that CYP3A4 is the isozyme that metabolises cisapride

Front 42

what is oxidation on heteroatoms by cytochrome P450?

Back 42

-can occur on nitrogen or sulfur atoms

Front 43

What is the experiment used as an example of oxidation on heteroatoms? How was it significant?

Back 43

-N-oxidation of procainamide by human liver microsomes
-also done under N2 air and the presence of cimetidine
-also boiled to see if it's an enzymatic reaction, it is
-N2 not an inhibitor of enzyme, displaces O2 which makes the inhibitor not work, safer than CO

Front 44

What are reductions done by cytochrome p450? Example?

Back 44

-can occur when O2 tension is low
-may lead to formation of free radicals
-halothane dehalogenation (oxidation vs reduction)
-uses aliphatic hydroxylation, but 90% oxidative
-can cause liver damage

Front 45

-What are desaturations by cytochrome p450? Example?

Back 45

-direct conversion of single bond to double bond
-probably occurs via free radicals
-desaturation of valproic acid

Front 46

What is flavin monooxygenase (FMO)?

Back 46

-flavin containing enzyme
-microsomal, requires O2 and NADPH
-catalyses oxidations on nitrogens and sulfur atoms (never carbon)
-products may be identical to those produced by CYPs

Front 47

What experiment is a good example of flavin monooxygenase? How is it significant?

Back 47

-S-oxidation of thiobencard by striped bass liver microsomes
-thiourea was used as an FMO inhibitor and aminobenzotriazole was used as a ABT and CYP inhibitor
-FMO is sensisitve to heating
-metabolism wasn;t affectede by CYP inhibitor but by FMO inhibitor

Front 48

What are molybdenum hydroxylases?

Back 48

-include aldehyde oxidase (AO) and xanthine oxidase (XO)
-contain molybdenum, flavin, and iron in active site
-cytosolic, water is oxygen donor
-generally catalyse oxidations adjacent to a nitrogen or sulfur
-also CYP dependent

Front 49

What experiment is a good example of molybdenum hydroxylases? How is it significant?

Back 49

-oxidation of 6-oxypenciclovir by partially purified hepatic aldehyde oxidase
-menadione/isovanillin uses as aldehyde oxidase inhibitor and allopurinol as an xanthine oxidase inhibitor
-showed that xanthine oxidase is dependent on aldehyde oxidase

Front 50

What is alcohol dehydrogenase (ADH)?

Back 50

--distributed mostly in liver, also kidney and lung (cytosolic)
-cofactor is B-nicotinamide adenine dinucleoside (NAD+)
-oxidizes alcohols to aldehydes (reversible)

Front 51

What is aldehyde dehydrogenase (AIDH)?

Back 51

-distribution is mostly in liver
-cofactor is NAD+
-oxidizes aldehydes to carboxylic acids (irreversible)

Front 52

What experiment is a good example of alcohol and aldehyde dehydrogenase? What is significant about it?

Back 52

-pyrazole was used as a ADH inhibitor
-disulfiram was used as a AIDH inhibitor
-ADH is faster that ADIH
-so AIDH is dependent on ADH for reaction to complete
-so liver damage can occur if AIDH doesn't react

Front 53

What is prostaglandin H synthase (PHS)?

Back 53

-catalyses conversion of arachidonic acid to prostaglandin H2 (PGH2) via prostaglandin G2 (PGG2)
-extrahepatic (renal medulla)
-limited role in metabolism of xenobiotics

Front 54

What experiment is a good example of prostanglandin H synthase? What is signifcant about it?

Back 54

-renal metabolism of acetaminophen (APAP)
-indomethacin used as a PHS inhibitor

Front 55

How is alcohol dehydrogenase a reductive enzyme? Example?

Back 55

-catalyses reduction of aldehyde to alchohol
-suhc as reduction of chloral to trichloroethanol

Front 56

What is mammalian aldo-keto reductase (liver)? example?

Back 56

-reduction of a ketone to secondary alcohol
-reduction of naltrexone

Front 57

What are bacterial reductases (intestinal)? Example?

Back 57

-reduction of a azo group to an amino group
-reduction of sulfasalazine

Front 58

What enzymes conduct hydrolyses?

Back 58

-epoxide hydrolase (EH)
-esterases
-amidases

Front 59

What is epoxide hydrolyase (EH)? Example?

Back 59

-catalyses addition of water to epoxides forming dialcohols
-microsomal and cytosolic forms known
-widely distributed in the body (liver, kidney, lungs, intestines, etc)
-detoxifies potentially reactive epoxides
-hydrolyses of safrole epoxide

Front 60

What are esterases? Example?

Back 60

-catalyses the hydrolyses of esters to carboxylic acids and alcohols
-widely distributed in body (nicrosomes, cytosol, and blood plasma)
-occurs durign hydrolyses of aspriin to salicyclic acid

Front 61

What are amidiases? Example?

Back 61

-catalyse hydrolysis of amides to carboxylic acids and amines
-distribution somewhat more restricted than esterases
-occurs during hdyrolysis of procainamide to p-aminobenzoic acid

Front 62

What are internal factors that affect drug metabolism?

Back 62

-species/strain
-age
-gender
-hormonal affects
-genetic polymorphisms

Front 63

How does the species/strain affect drug metabolism? What are the phase I enzymes for various species? Phase II enzymes?

Back 63

-it can affect phase I or phase II pathways
-Phase I enzyme in humans in CYP3A4
-Phase I is male rats is CYP3A1/3A2
-Phase I in female rats is CYP3A9
-Phase II for cats is UGT1A6 deficiency and NAT1/NAT2 deficency in dogs
-phase II for pigs is SULT1A1
-strain-dependent differences most obvious in rodents

Front 64

What specific experiments are examples of how species/strain affects drug metabolism? Significance?

Back 64

-para vs ortho ainiline hydroxylation
-cats and dogs are ortho dominated
-ferrets are half and half
-mouse, rats, and gerbils are para dominant
-may be due to different shape in active sites of enzymes in different species
-hexobarbital-induced sleeping time in mice
-diiferent strains can metabolize hexobarbital faster than others

Front 65

How does age affect drug metabolism?

Back 65

-reduced biotransformation in fetal and newborn animals
-metabolism generally declines with age
-infants use CYP3A7 then CYP3A4 when grown up

Front 66

What experiment is a good example of age affect drug metabolism? significance?

Back 66

-effect of age on liver microsomes from female rats
-metabolism of certain enzymes slows age rats age

Front 67

How does gender affect drug metabolism? What experiment is a good example? Significance?

Back 67

-xenobiotic metabolism is generally similar in humans
-gender-related differences very common in rodents
-dealkylation of ethylmorphine
-metabolism differences can be seen in rats but not in other species

Front 68

How do hormonal effects affect drug metabolism? Experimental examples? significances?

Back 68

-usually very complex
-diabetes can be involves
-effect of streptozotocin (STZ)-induced diabetes on metabolism in male rats
-metabolism decreases even though CYP concentration stays the same
-other endocrine organs (adrenals, thyroid, etc.) can decrease metabolism if removes and can restore activity by repleacement therapy with appropriate hormones
-pregancy can be involved
-effect on metabolism in rats
-certain enzyme metabolism is decreased while other are increased

Front 69

How do genetic polymorphisms affect drug metabolism?

Back 69

-N-acetyltransferases (NATs) polymorphisms are well recognized
-different ethnic groups have a higher percentage of slower metabolism than other ethnicities
-cytochrome p450 is highly polymorphic
-glutathione S-transferase deficiency may be assoiciated with increased risk in certain cancers
-increased toxicity of irinotecan in UGT1A1-deficient individuals

Front 70

How do environmental conditions affect drug metabolism? Experimental example? Significance?

Back 70

-modification of natural circadian rhythms
-effect of light on CYP activity in rats
-increase sleep time leads to decreased metabolism and vice-versa

Front 71

What other factors affect drug metabolism other than itnernal or environmental factors?

Back 71

-enzyme inducers and inhibitors
-dietary diefiencies can have unpredictable effects
-such as: starvation (CYP2E1 induction), protein/fat, carbohydrates, vitamins, and minerals
-disease status of liver such as infections/tumors, and chronic ehtnaol abuse which decreases drug metabolism

Front 72

What is glucuronidation?

Back 72

-substrate is conjugated with glucuronic acid
-glucuronide conjugates are usually non-toxic
-common phase II pathway (depends on species)
-cofactor is readily available
-enzymes have wide substrate specificity

Front 73

What is the mechanisms of the reaction of glucuronidation?

Back 73

-involves a microsomal enzyme (very tightly membrane-bound) known as UDP-glucuronosyltranserfase (UGTs)
-isozymes are known (e.g. UGT1A1)
-reaction proceeds with inversion of stereochemistry
-cofactor is uridine-5'-diphospho-a-D-glucuronic acid (UDPGA)
-occurs on oxygen, nitrogen, sulfur (rare) and even carbon (very rare)

Front 74

What specific reactions can occur during glucuronidation? Examples of each?

Back 74

-O-glucuronidation can occur with alcohols, ohenols, and carboxylic acids (such as 1-naphthal)
-N-glucuronidation can occur with amines, amides, and sulfoamides (such as desipramine)
-S-glucuronidation (rare) can occur with thiol (-SH) (such as methimazole)
-C-glucuronidation (very rare) can occur on alkynes or 1,3 dicarbonyl groups (such as phenylbutazone)

Front 75

What is sulfation?

Back 75

-substrate is conjugated with sulfate ion (more polar)
-sulfate conjugates are usually non-toxic
-can compete with glucuronidation (depends on substrate and species

Front 76

What is the general reaction for sulfation?

Back 76

-requires cytosolic enzyme known as sulfotransferase (SULTs)
-isozymes are known (e.g SULT1E1-estrogen sulfotransferase)
-cofactor is 3'-phosphoadenosine-5'phosphosulfate (PAPs)
-such as acetaminophen
-occurs primarily with phenols and to a lesser extent with albohols

Front 77

What is acetylation? What is the general reaction? Example?

Back 77

-substrate is conjugated with an acetyl (COCH3) group (less polar)
-cytosolic enzymes known as N-acetyltransferases (NAT1 and NAT2)
-cofactor is acetyl coenzyme A (CH3CO-S-CoA)
-occurs most commonly with amines (esp. if attached to an aromatic ring)
-genetic polymorphism exists (slow vs. fast acetylators)
-such as acetylation of sulfamethazine

Front 78

What is methylation? What is the general reaction? example?

Back 78

-substrate is conjugated with a methyl group (CH3) (less polar)
-relatively rare for drugs, more common for endogenous substances
-involves enzyes nown as methyl transferases (cytosolic)
-cofactor is S-adenosylmethione (AdoMet or SAM)
-occurs on oxygen, nitrogen, or sulfur
-such as O-methylation of tea polyphenols by human placental catechol O-methyltransferase

Front 79

What is amino acid conjugation? What is the general reaction? Example?

Back 79

-substrate is conjugated with an amino acid (more polar)
-relatively minor pathway for most drugs
-species-dependent
-catalyzed dequentially by CoA synthetase and amino acid N-acryltransferase (mitochondrial)
-cofactors include ATP and coenzyme A
-occurs only with carboxylic acids
-such as glycine conjugation of salicyclic acid to salicycluric acid (70% total)
-amino acid conjugation of vaproic acid (VPA)

Front 80

What is glutathione conjugation? General reaction? examples?

Back 80

-substrate is conjugated with glutathione (less reaction and more polar)
-catalysed by glutathione S-transferase
-cytosolic and isozymes are known
-important detoxification pathway for epoxides and quinone
-also metabolizes acetaminophen for detoxification of NAPQI

Front 81

What is interorgan metabolism of glutathione conjugates?

Back 81

-conjugate probably formed in liver
-metabolized to cystein conjugate in bile duct/intenstines (y-glutamyltranspeptidase removes the glutamic acid and cysteinylglycine dipeptidase removes the glycine)
-cystein conjugate may be converted to an N-acetylcysteine conjugate (mercapturic acid)
-elimination or further metabolism may occur in the kidneys such as cystein and/or N-acetylcystein conjugates may be elimnated by urine or may be converted to a thiol conjugate for further metabolism

Front 82

What is the anatomy/physiology of the liver?

Back 82

-hepatocytes radiate out in "cords" from central veins
-extraction of solutes from sinusoidal blood
-secretion of drugs/metabolites from liver (blood vs bile)
-barrier of movement exists b/t blood and bile
-blood goes through sinusoidal membrane
-bile goes through canalicular membrane

Front 83

What is the function of bile?

Back 83

-digestion of lipids
-excretion of bilirubin(deggradation of hemeglobin) , excess cholesterol, etc.

Front 84

What is the composition of bile?

Back 84

-made up of water, ions, bile salts, GSH, phase II metabolites, etc.
-similar electrolyte composition to plasma, but essentially protein-free

Front 85

What is the flow of bile?

Back 85

-solute transport (biliary excretion of bromosulfophthalein) is actually active transport since it can become saturated
-osmotic movement of water through aqua porin channels
-concentrated/stored in gall bladder
-canalicular contractions are involved to push bile through channels

Front 86

What is choleresis? Cholestasis?

Back 86

-increase in bileflow
-decrease in bile flow
-can lead to drug-drug interactions (phenobarbital)

Front 87

What are class A compounds secreted in bile?

Back 87

-glucose, Na, K, Cl, Hg, Cs, Th, Co

Front 88

What are class B compounds secreted in bile?

Back 88

-bile salts, bilirubin, phase II conjugates, creatinine, oubain, Pb, As, Mn

Front 89

What are class C compounds secreted in bile?

Back 89

-sucrose, inulin, albumin, phosphates, phospholipids, Zn, Au, Fe, Cr

Front 90

What factors can affect biliary secretion?

Back 90

-polarity
-molecular weight threshold (species-dependent)
-such as effect on excretion of biphenyl/chorobiphenyls in rats
-higher molecular weights go to feces (from bile)

Front 91

What hepatocyte transporters are in the basolateral membrane?

Back 91

-sodium-taurocholate cotransporting polypeptide (solute carrier)
-organic anion transporting polypeptides (solute carrier)
-organic cation transporters (solute carrier)
-multidrug resistance-assoicated protein 3 (ATP binding cassette)

Front 92

What hepatocyte transporters are in the canalicular membrane?

Back 92

-bile salt export pump (solute carrier)
-p-glycoprotein (ATP bindng cassette)
-multidrug resistance-assoicated proteien 2 (ATP binding cassette)

Front 93

What are biliary metabolites?

Back 93

-undergo fecal excretion or intestinal reabsorption
-outcome depends on polarity and MW of compounds
-further metabolism is possible (such as GSH conjugates and glucuronides and sulfates)
-extent of enterohepatic recycling can alter half-life of drug in body (increased is enhanced toxicity while decreased reduces toxicitiy
-biliary excretion can be altered by choleresis, cholestasis, liver disease, and broad spectrum antibiotics

Front 94

What techniques/examples are used with bile?

Back 94

-bile duct cannulation (biliary and urinary excretion of desmethylnaproxen in rats)
-canalicular membrane vesicles (carrier-mediated uptake of quinoline antibiotics)