Phase 2 Metabolism

• Glutathione-S-transferases (GSTs) are mainly soluble enzymes in the cytosol.
• They are composed of homo- or heterodimers with a molecular weight of approximately 25,000.
• GSTs can make up to 10% of total cytosolic protein in a hepatocyte.
• They conjugate reduced glutathione to electrophilic compounds through the sulfur of a sulfhydryl group.
• GSTs are found in most human tissues and exist in a large number of different isoforms.
• They also function as intracellular binding proteins.

GST Polymorphisms

• Genetic polymorphisms have been identified in three GST isoforms: GSTM1, GSTT1, and GSTP1.
• GSTM1 and GSTT1 polymorphisms result in a loss of enzyme activity.
• A GSTM1 loss of activity affects 50% of Europeans, detectable by measuring trans-stilbene oxide conjugation in lymphocytes.
• GSTM1 polymorphism was established by 1988 due to its large deletion, easily detected through various approaches.
• GSTP1 polymorphism results in a small change in catalytic activity that is not very impactful on enzyme activity.

GSTM1 Polymorphism

• GSTM1 detoxifies benzo[a]pyrene diol epoxide, which becomes toxic when metabolized by CYP1A1.
• Approximately 50% of white Europeans lack GSTM1 enzyme activity.
• GSTM1-deficient individuals are homozygous for a large deletion.
• About 1% of Saudi Arabians have a gene duplication of GSTM1.
• White Europeans completely lack the M1 fragment, while some individuals have two copies of the M1.
• Trans-stilbene oxide is a specific substrate for the Mu 1 isoform of GST.

GSTT1

• GSTT1 belongs to the Theta class of GSTs.
• It prefers small organic molecules like dichloromethane, which is unusual for GSTs.
• Approximately 10-20% of Caucasians lack GSTT1 due to a deletion.
• Volunteers have decreased activity to 2-bromoethane, measured by activity in erythrocytes (P3).
• Genotyping confirms gene deletion in some individuals.

Inter-Population Variation of GSTT1 and M1 Polymorphisms

• GSTM1 null: 50% of Europeans, 30% of African Americans, 81% of Polynesians, 48% of Japanese.
• GSTT1 null: 15% of Europeans, 22% of African Americans, 60% of East Asians.

N-acetyltransferases (NATs)

• NATs can transfer an acetyl group to hydroxyl groups.
• Human NATs are found in the cytosol.
• They are small proteins produced by adjacent intronless genes.
• NAT1 is expressed in most tissues, while NAT2 is mainly in the liver and intestine.
• Both enzymes have distinct but overlapping substrate specificities.
• NATs are not inducible.

NAT2 Polymorphism

• In the 1950s, slow metabolism of isoniazid was linked to peripheral neuritis.
• In 1991, NAT2 was confirmed to metabolize isoniazid.
• A defect affects approximately 50% of Europeans and is inherited as an autosomal recessive trait.
• Individuals with normal activity are fast acetylators.
• Slow acetylators can be identified by phenotyping (e.g., with caffeine) or genotyping.
• N-acetylation does not influence the pharmacological activity of caffeine.
• The wild-type NAT2 in humans is NAT2*4.
• No small or large deletions are known for this gene.
• NAT25 has reduced activity, while NAT26 and NAT2*7 have effectively no activity.
• NAT2*5's reduced activity is due to: C341T (Ile114Thr), C481T, and A803G (Lys268Arg).
• Polymorphisms with low or no activity: NAT6 (C282T , G590A (Arg197Gln)) and NAT7 (G857A (Gly286Glu)) are very detrimental to activity.
• Homozygotes for *6 and *7 alleles have been described as ultra slow acetylators.

NAT2 Genotyping

• Genotyping involves identifying individual variant alleles through diagnostic reactions.
• Slow acetylators include genotypes such as *5/*5, *5/*6, *6/*6, *5/*7, *6/*7, and *7/*7.

Inter-population Variation in Frequency of NAT2 polymorphism

• 50% of Europeans are slow acetylators.
• 10% of East Asians are slow acetylators.
• The *7 variant allele is more common in East Asians but *5 and *6 in Europeans.

Glucuronidation

• Glucuronidation is a widely used Phase 2 reaction performed by UDP-glucuronosyltransferases (UGTs).
• UDP-glucuronic acid is required as a cofactor.
• Endogenous compounds (steroids, vitamins, bile acids, and bilirubin) also undergo conjugation by UGTs.

Polymorphisms in UGT Genes

• UGT1A1 (bilirubin metabolizing form) polymorphisms lead to rare inborn errors of metabolism (Crigler-Najjar syndrome) or Gilbert's syndrome.
• Crigler-Najjar syndrome results in a complete absence of the enzyme.
• Gilbert's syndrome is associated with higher than normal plasma bilirubin.
• In newborns, haem is converted to insoluble bilirubin, requiring glucuronidation for excretion.
• Accumulation of bilirubin becomes toxic to organs and tissues.
• Gilbert syndrome results in a small increase in plasma bilirubin with no adverse clinical consequences.

Gilbert Syndrome

• The most common defect in Caucasians is (TA)7TAA in the upstream regulatory region instead of (TA)6TAA, affecting transcription levels.
• Some Asians have an amino acid substitution in exon 1, resulting in the same phenotype (lower catalytic activity).
• This polymorphism impacts irinotecan metabolism.
• Either dose adjustment or alternative drug to be prescribed is recommended
• Less UGT1A1 transcript is formed.
• In Japanese individuals, protein concentration remains the same, but catalytic activity is less efficient.

UGT2B7 Polymorphism

• UGT2B7 glucuronidates various drugs, including morphine and NSAIDs.
• A common polymorphism (C180T) results in a His268Tyr substitution
• Complete linkage disequilibrium with C-161T polymorphism close to promoter region.
• C alleles are denoted *1, T alleles are denoted *2.
• TT is suggested to be associated with increased morphine 6- glucuronidation in vivo.

Sulfotransferase Polymorphisms

• The Main sulfotransferase where polymorphism has been well studied is SULT1A1.
• Polymorphisms include copy number variation and variation in the 3'-untranslated region regulated by miRNA.

Copy Number Variation of SULT1A1

• Different populations contain varying copies of genes in SULT1A1.

The untranslated region (3'-UTR)

• The 3'-UTR: the section of mRNA that immediately follows the translation termination codon.
• Contains elements which regulate gene expression post-transcriptionally
• Contains binding sites for regulatory proteins and microRNA's (miRNAs)
• miRNA binding to 3'-UTR can reduce gene expression by inhibiting translation or by mRNA cleavage​.

Yu et al, 2010 study

• Displayed 3'-end polymorphism affecting SULT1A1 activity.
• CC is the homozygous wild-type.
• Reduced SULT1A1 activity from CC to CT and furthermore a significant difference between CT and TT.
• Enzyme activity is a small change but is still measurable.
• C973T is at 3'-end of gene in non-translated region.
• The allele frequency of T is 0.44 so approx. 20% of white US population homozygous

Polymorphism in 3'UTR Region

• At bp 973 in this region, if constructs containing C will show a lower extent of miRNA binding and hence more luciferase activity; these patients have measurable activity.
• If this bp has a T construct, it will have more efficient binding to miRNA-631 binding, hence less luciferase activity; these patients have reduced activity.

Regulation of SULT1A1 Expression by miRNA

• miR-631 downregulates SULT1A1 expression more robustly in subjects carrying 973T than in those with C allele.
• The gene expression for SULT1A1 was under the control of this microRNA
• Co-transfection with a miR-631 inhibitor in MCF-7 cells resulted in increased SULT1A1 protein expression.

Thiopurine-S-methyltransferase (TMPT)

• It metabolizes thiopurine drugs, suppressing DNA/RNA and protein synthesis, with cytotoxic and immunosuppressive properties.
• S-adenosylmethionine donates a methyl group.
• Deficiency is associated with high levels of cytotoxic thioguanine nucleotide metabolites.
• ADRs of thiopurines: myelosuppression and death.
• 0.3% of Caucasians lack this enzyme.
• 1/300 Europeans lack this enzyme.
• Lower activity observed in heterozygotes.
• It is important to genotype individuals undergoing treatment with mercaptopurine or azathioprine (a 6-MP precursor) for autoimmune diseases.

TMPT Polymorphisms

• Main variant alleles are associated with amino acid substitutions.
• Found in 1996 in Evans and Weinshilboum laboratories.
• TMPT2 alleles are less common than TMPT3 alleles.
• The TMPT*3 alleles are more complex, there are 2 amino acid changes.
• G460A (Ala154Thr) and A719G (Tyr240Cys) polymorphisms lead to a loss of activity.
• TMPT3 alleles are made of TMPT3A, TMPT3B, TMPT3C.
• NUDT15 increases the risk of thiopurine-induced leukopenia, but TMPT polymorphisms improving use of mercaptopurines is unclear at the present.

Catechol-O-methyltransferase Polymorphism

• COMT is involved in catecholamine metabolism.
• Involved in NA to normetanephrine
• Metabolizes some drugs such as L-DOPA.
• A common genetic polymorphism in Codon 158 with a G to A polymorphism results in Val and Met substitution.
• The Met form enzyme is more thermolabile than Val and associated with lower activity.

DNA Methyltransferases

• Aberrant alterations in methylation status of CpG dinucleotides in cytosine residues occur in promoter enhancer regions of CYP1B1 in prostate cancer.
• Activation of CYP1B1 via the AhR/RNT pathway requires recognition of the dioxin response element (DRE), also called xenobiotic response element (XRE).
• Benign prostatic hyperplasia and prostate cancer reveals increased levels of latter linked with demethylation of CpG islands in DRE observed in prostate cancer.
• The transcription factor Sp1 binding site GGGCGG was demethylated in prostate cancer tissues but remained methylated in BPH.
• Hypomethylation of sites in the promoter and enhancer regions of CYP1B1 allows binding transcription factors and promotes expression of the CYP1B1 gene in prostate cancer.

Questions on drug metabolism, GSTs (Glutathione-S-transferases), NAT2 alleles, and UDP-glucuronosyltransferases (UGTs). Explore the function, polymorphisms, and characteristics of these important enzymes involved in detoxification and drug metabolism.