Phase 2 Reaction

Phase II Reactions

conjugation reactions:

– sulfate conjugation
– glutathione conjugation
– glucuronidation
– acetylation
– methylation
– amino acid conjugation

Sulfate Conjugation

• makes sulfate ester

• catalyzed by a dozen of sulfotransferases (SULTs)

• uses PAPS as a high energy sulfonate donor

SULT

• locates in cytosol of liver, GI mucosa and kidney

• modulate the activities of steroid hormones, nuerotransmitters, toxicants and drus

• different sulfotransferases from species as well as related forms of sulfation enzymes from different species, rats and humans, differ in thir substrate specificities and tissue distributions

PAPS

• 3’-phospho-5’-adenylyl sulfate

• high energy sulfonate donor

Products of Sulfate Conjugation

1. Sulfonated molecules are highly soluble in water and easily

excreted from the organism.

2. Electrophilic cation intermediates formation: Sulfate group is

electron-withdrawing and may be cleaved off heterolytically in

some molecules, leading to an electrophilic cation (+).

– an electrophile is a chemical species that forms bonds with

nucleophiles by accepting an electron pair.

DNA adduct

• Some sulfation reaction intermediates can spontaneously rearrange to release

sulfate and generate a reactive electrophilic cation molecule. Such electrophilic

molecules can react with DNA to form adducts.

• DNA adduct is a chemical covalently bond to a piece of DNA. This can interfere with

DNA replication, leading to toxicity and mutations.

– An electrophile is a chemical species that forms bonds with nucleophiles by

accepting an electron pair.

– An electrophilic cation is inherently reactive and can form covalent binding to

nucleophilic components, such as proteins, nucleic acids, and lipids.

– responsible for certain mechanisms of carcinogenicity and tumor

formation.

Sulfation of Methyleugenol

• DNA adduct could be start of carcinogenesis

• found as natural consituent in many plants

Glutathione

• tripeptide that is one of the most important molecules in the
  cellular defense against toxic compounds

• It is found in most cells but is especially abundant in the red blood cells
  and in the liver (5mM)

Glutathione functions

• antioxidant

• forms glutathione conjugates to increase water solubility of the target substance

alpha peptide bond

covalent bond formed between two amino acids: They are
amide linkages formed between the carboxyl group (COOH) of one amino acid and the amino group (NH2) of another, releasing a molecule of water (H2O).

gamma peptide bind

first peptide bond that forms Glutathione is formed between the gamma-carboxyl group of the Glu side chain and the alpha-amino group of Cys

y-glitamyltranspetidase

only enzyme that can hydrolyze glutathione and are found on the surface of cells

Glutathione Conjugation

via glutathione s-transferases (GSTs) results in the formation of a glutathione-S conjugate

Glutathione Conjugation in Liver

1. Xenobiotics conjugated with glutathione include aromatic, aliphatic, heterocyclic and alicyclic epoxides, halogenated aliphatic and aromatic compounds, aromatic nitro compounds, unsaturated aliphatic compounds and alkyl halides

2. Reaction of the conjugation of the reduced form of glutathione (GSH) to xenobiotics is catalyzed by glutathione-S-transferases (GSTs)

3. GSH’s net negative charge and overall hydrophilicity greatly increases the aqueous solubility of the lipophilic moieties with which it becomes conjugated

Location of Glutathione

1. Mainly in cytosol

2. Some are found in the endoplasmic reticulum, i.e. microsomal

enzymes. Others are found in mitochondria

• Some are soluble proteins, while others are membrane bound

• Both types, the soluble GSTs and microsomal membrane bound

enzymes, exhibit a broad tissue distribution: they are found in liver,

kidney, brain, lung, heart, pancreas, small intestine, prostate, spleen,

and skeletal muscles

Glutathione and Excretion

1. Relatively more polar glutathione conjugates are eliminated into the bile.

2. Its molecular size (m.w. 307) ensures that its conjugated products are preferentially secreted via the biliary system which selects molecules of molecular weight greater than 300 to 500 according to the species, 325 ± 50 for rats and 500 ± 50 for humans.

3. In mammals, GSH conjugates are often further metabolized by hydrolysis and N-acetylation, either in the gut or in the kidney, to give N-acetylcysteinyl conjugates known as mercapturic acids, which are excreted in the urine.

increases bile and urinary excretion

Mercapturic Acid Formation

• For most compounds, mercapturic acid formation is a detoxication pathway, but the

precursor CysS-conjugates may undergo bioactivation to reactive intermediates

(Hashmi et al.1992; Anders and Dekant1998).

• The metabolites of endogenous substrates formed by the mercapturic acid pathway

may play a role in respiratory diseases, cancer, neurological disorders, and

cardiometabolic diseases that contribute to the pathophysiological development of

chronic inflammatory and metabolic disorders (

Cystein Conjugate and β-lyase

• β-lyase metabolizes S-cysteine conjugates via both transamination and β-elimination.

• The end products: pyruvate, ammonia, and a sulfur-containing fragment

• The sulfur-containing intermediates are chemically reactive, and particularly toxic to kidney mitochondria.

• The presence of β-lyase in the kidney makes some cysteine conjugated substances nephrotoxic.
  • Example of the industrial chemical 4-bromobenzene  Cysteine conjugate of 4-bromobenzene

• can make aryl thiols that are very toxic

Toxifixation by β-Lyase

• β-Lyase is present in the cytosolic fraction of rat liver and kidney and also in the

microflora of the gut.

• Since thiols may be toxic and are more lipophilic than their cysteine conjugate

precursors, β-lyase reactions generally constitute a toxification pathway.

Glutathione circadian variation

A trend toward increasing GSH concentration at 9:00 AM and decreasing
concentration at 1:00 AM

Glucuronidation Reaction

• UDP-glucuronic acid (UDPGA): Activated glucuronic acid donor, as uridine

diphosphate glucuronic acid

• UGT: UDP-glucuronosyltransferase (enzyme)

• R–X–glucuronide: Conjugated (more water-soluble) product

• UDP: Uridine diphosphate (leaving group)

Glucuronidation

a major metabolic reaction, and mainly takes place in the
liver, for disposal of a variety of endogenous (such as Thyroid hormone) and
exogenous substrates (such as PCBs). It involves the transfer of glucuronic
acid from the cofactor UDP-glucuronic acid to functional groups, in
particular to nucleophilic functional groups, including hydroxyl groups (-
OH), amino groups (-NH2), or carboxyl groups (-COOH).

• PCBs/TCDDs are inducers of enzymes involved in glucuronidation.

• Unlike sulfotransferases (SULTs), UGTs have low specificity and high capacity.

• Glucuronic acid conjugation substantially increases the hydrophilicity of a

substrate enhancing its excretion.

UDP-GA Formation

formed in cytosol from glucose-1-phosphate in 2-step reaction:

• 1st step is addition of uridine diphosphate (UDP) to glucose-1-phosphate

catalyzed by UDPG pyrophosphorylase

• 2nd step is conversion of UDP glucose to UDP glucuronic acid catalyzed by

UDPG dehydrogenase; oxidation of the substrate C6 alcohol into the

corresponding carboxylic acidx

Enzymes for Glucose to Glucuronide

1. Hexokinase:

– Glucose + ATP  Glucose-6-P + ADP

2. Phosphoglucomutase:

– Glucose-6-P glucose-1-P⇌

3. UTP—glucose-1-phosphate uridylyl transferase, also known as glucose-1-phosphate

uridylyl transferase (or UDP–glucose pyrophosphorylase), is an enzyme involved in

carbohydrate metabolism. It synthesizes UDP-glucose from glucose-1-phosphate and

UTP:

– glucose-1-phosphate + UTP UDP-glucose + pyrophosphate⇌

4. UDP-glucose 6-dehydrogenase is a cytosolic enzyme, primarily occurs in the liver, also

found in kidneys and brain:

– UDP-glucose + 2 NAD+  UDP-glucuronic acid + 2 NADH + 2 H+

5. UDP-glucuronyltransferases (UGTs):

– UDP-glucuronic acid + substrate  Glucuronide

Formation of UDP-GA and Conjugation

Billibrubin

lipid-soluble, orange-yellow, metabolite produced in the
liver by the breakdown of hemoglobin. It is practically insoluble in
water, mw 584.66. Glucuronidates billirubin is excreted in bile.

Billirubin toxicity

• Bilirubin (unconjugated and free bilirubin not bound by albumin) causes oxidative damage at the cellular level, causing oxidative stress through dysfunctions of endoplasmic reticulum, mitochondria, and plasma membrane.

• The most concerned biological effects of bilirubin are its harmful effects on the CNS of NBIs when it reaches high serum levels.

• In newborns the cell injuries could cause bilirubin encephalopathy and irreversible neurologic dysfunction, including hearing loss and even death. The threshold of bilirubin toxicity is lower in preterm or premature newborns.

• However, molecular biology studies in adults have evidenced that bilirubin is a cytoprotective antioxidant.

• Low to moderately high bilirubin levels account for a potent antioxidant.

• However, paradoxically, once certain threshold is exceeded, it is harmful for the developing brain.

• Different studies have demonstrated that oxidative stress is a key mechanism in bilirubin induced neurotoxicity.

• Bilirubin is lipid-soluble, passes through the membranes, and is incorporated to cells and organs. In the setting of intrauterine life, this characteristic allows bilirubin to cross the placental barrier from the fetus to the mother.

Billirubin threshold

• Bilirubin in blood is mostly bind to albumin. Only free indirect bilirubin (FIB) can pass through blood-brain barrier and cell membranes.

– Preterm newborns have lower albumin binding capacity.

– Conditions like hypothermia, hypoxia, acidosis, and asphyxia favor

neurotoxicity by breaking the binding and generating FIB.

– Certain drugs like ceftriaxone and ibuprofen compete with bilirubin for

albumin binding

– High lipid intake, > 1.5 g/kg, significantly reduces the bilirubin-albumin binding capacity and increases FIB levels in PNBIs born at less than 28 weeks of gestational age.

• Physiological hemolysis caused by oxidative stress due to the change from

placental to pulmonary oxygenation.

N-Acetylation

• Enzyme: N-acetyltransferases (NAT), two isozymes NAT1 and NAT2

• Cofactor: acetyl-coenzyme A (acetyl CoA)

• N-acetylation is a major route of biotransformation for xenobiotics

containing an aromatic amine (R-NH2)

• Unlike other Phase II reactions, acetylation masks an amine with

nonionizable group and are less water soluble than the parent

compound

• present in mercapturic-acid conjugates

N-acetyltransferases

• Acetylation is catalyzed by cytosolic N-acetyltransferases with acetyl

coenzyme A as the required cofactor.

• This reaction is particularly important for compounds such as

aromatic amines

• The enzymes are found in most mammals with the exception of dogs

(and foxes), which are unable to acetylate xenobiotics.

Acetaminophen in cats and dogs

• Acetaminophen (APAP)-glucuronide was the principal metabolite excreted in the urine of dogs; its fraction of the total metabolites excreted in urine remained constant at the three dose levels

• Cat is at increased risk from APAP exposure because of impaired glucuronidation. In cats, APAP-sulfate was the major metabolite in urine at all three dosage levels, but the fraction of the total urinary metabolites represented by APAP-sulfate decreased as the dosage increased, suggesting saturation of its sulfate conjugation pathway.

Methylation

adding a –CH3 group, does not dramatically alter the solubility of
substrates and results either in inactive or active compounds, requires the co-factor S-adenosylmethionine (SAM)

Methylation of Arsenics

• AsIII is most toxic, in terms of acute toxicity.

• As methyltransferase (AS3MT), cofactor SAM

• Monomethylarsonic acid (MMAv) to dimethylarsinic acid (DMAV)

– These two organic arsenic metabolites are not readily excreted.

• Thioredoxin (Trx) is a class of small redox proteins; AS3MT: arsenite

methyltransferase (human)

glycine

most common amino acid in amino conjugation

Hippuric Acid formation

the conjugation involves initial formation of a benzoyl acyl-CoA thioester that is then conjugated with glycine. this is done with benzoic acid.

Toulene

Solvent in glues and household products.Biotransformation primarily involves liver enzymes, particularly cytochrome P450, oxidizing it to benzyl alcohol, then further to benzoic acid, which is then conjugated with glycine to form hippuric acid, the primary urinary metabolite.

cholic acid

major bile salt with conjugates of glycine and taurine that facilitates the absorption of lipids and lipid-soluble vitamins

Species variations in amino acid conjugates in bile

• In most species, including humans, primates, dogs, and rats, bile acids

can be conjugated with glycine and taurine.

• Rabbits and pigs form predominantly glycine-conjugated metabolites.

• Cats have limited ability to synthesize glycine-conjugated bile acids. In

cats, bile acids are almost exclusively (over 90%) taurine conjugates.

• The needs for taurine can be sufficiently synthesized by humans and

dogs, but cats have limited capacity to synthesize taurine.

• Taurine deficiency can lead to various disorders, such as blindness and

dilated cardiomyopathy

• While dogs utilize glycine conjugation, they are not inherently deficient in it; rather, they have a greater taurine synthetic ability and can adapt to using glycine for bile acid conjugation, unlike cats.

taurine deficiency

• Endogenously produced bile acids are mostly synthesized in the liver: they are taurine or glycine conjugates in humans.

• High capacity for taurine conjugates in cats and dogs.

• Both human and dogs can synthesize taurine, but not cats.

• Taurine deficiency:

– Mitochondria diseases

– Impairing the vision, retinal cells degeneration, such as feline taurine retinopathy.

– Central retinal degeneration (FCRD).

– Dilated cardiomyopathy (DCM), weakening of the muscle cells in the heart

– Taurine is a component of bile salts, and its deficiency may cause digestive

disturbances.

– Studies suggest that taurine deficiency might be a driver of the aging process.