Phase 2 metabolism Flashcards
What are Glutathione-S-transferases (GST)?
Mainly soluble enzymes found in cytosol
GST consist of homo or heterodimers of subunits with Mr. approx. 25,000 and may constitute up to 10% of total cytosolic protein in a hepatocyte.
What is the primary function of GST?
Conjugate reduced glutathione to electrophilic compounds through sulfur of sulfhydryl group
GSTs are found in most human tissues and have a large number of different isoforms.
What are the GST polymorphisms described?
GSTM1, GSTT1, GSTP1
GSTM1 affects 50% of Europeans with loss of activity, while GSTP1 has a small change in catalytic activity that is not very impactful.
What is the effect of GSTM1 polymorphism?
Loss of activity affecting approximately 50% of white Europeans
Detected by measurement of trans-stilbene oxide conjugation in lymphocytes.
What is the significance of GST Mu gene cluster?
Shows fragments of 2 different restriction enzymes: EcoRI and Hind3
White Europeans M1 is completely missing, indicating gene deletion.
What does GSTT1 preferentially conjugate?
Small organic molecules
Unusual for a GST, which typically prefers larger substrates.
What is the inter-population variation for GSTM1 null?
50% of Europeans, 30% of African Americans, 81% of Polynesians, 48% of Japanese
These groups cannot react with substrates.
What are N-acetyltransferases (NAT)?
Enzymes that transfer acetyl groups to hydroxyl groups
Both human NATs are found in cytosol and are small proteins from adjacent intron-less genes.
What is the impact of NAT2 polymorphism?
Affects approximately 50% of Europeans
Inherited as autosomal recessive traits and linked to slow metabolism of isoniazid.
What is the wild-type for NAT2 in humans?
NAT2*4
Variants NAT25, NAT26, and NAT2*7 have reduced or no activity.
What is glucuronidation?
Most widely used phase 2 reaction
Carried out by UDP-glucuronosyltransferases (UGTs) using UDP-glucuronic acid as a co-factor.
What syndrome is associated with UGT1A1 polymorphism?
Crigler-Najjar syndrome
Results from complete absence of enzyme and can lead to toxic accumulation of bilirubin.
What is Gilbert’s syndrome?
Mild increase in plasma bilirubin without adverse clinical consequences
Most common defect in Caucasians is (TA)7TAA in the upstream regulatory region.
What does UGT2B7 metabolize?
Range of drugs including morphine and various NSAIDs
Common polymorphism (C180T) results in His268Tyr substitution.
What is the main sulfotransferase polymorphism studied?
SULT1A1
Involves copy number variation and 3’-untranslated region affecting regulation by miRNA.
What is the role of the 3’-UTR?
Regulates gene expression post-transcriptionally
Contains binding sites for regulatory proteins and microRNAs.
What is the effect of miR-631 on SULT1A1 expression?
Downregulates SULT1A1 expression
More robustly in subjects carrying the T allele at position 973.
What is the role of thiopurine-S-methyltransferase (TPMT)?
Metabolizes thiopurine drugs
Deficiency can lead to dangerously high levels of cytotoxic thioguanine nucleotide metabolites.
What percentage of Caucasians lack TPMT enzyme activity?
Approx. 0.3%
Important to genotype individuals undergoing treatment with thiopurines.
What does methionine donate?
Methyl group
What is the consequence of S-methylation of drugs when there is a deficiency of TPMT enzyme activity?
Dangerously high levels of cytotoxic thioguanine nucleotide metabolites
What are the adverse drug reactions (ADRs) of thiopurines?
Myelosuppression and death
What percentage of Caucasians lack the TPMT enzyme?
Approx. 0.3%
What is the approximate frequency of Europeans lacking the TPMT enzyme?
1/300
What is observed in heterozygotes regarding TPMT activity?
Lower activity
Why is it important to genotype individuals undergoing treatment with mercaptopurine or azathiopurine?
For autoimmune diseases
What are TMPT polymorphisms associated with?
Amino acid substitutions
In what year were TMPT polymorphisms identified?
1996
Which TMPT alleles are less common?
TMPT*2 alleles
What is the characteristic of TMPT*3 alleles?
More complex with 2 amino acid changes
What are the two amino acid changes in TMPT*3 alleles?
- G460A (Ala154Thr)
- A719G (Tyr240Cys)
What is the result of TMPT polymorphisms?
Loss of activity
What are the variants of TMPT*3 alleles?
- TMPT*3A
- TMPT*3B
- TMPT*3C
What does NUDT15 increase the risk of?
Thiopurine-induced leukopenia
Is the role of TMPT polymorphisms in improving the use of mercaptopurines clear?
Unclear at present
What is the role of COMT?
Catecholamine metabolism
What is the common genetic polymorphism in COMT?
Codon 158 G to A polymorphism
What substitutions occur due to the codon 158 G to A polymorphism?
Val and Met substitution
Which form of the COMT enzyme is more thermolabile?
Met form
What are aberrant alterations in methylation status associated with?
CpG dinucleotides in cytosine residues
What is targeted by DNMT in promoter enhancer regions?
CYP1B1 in prostate cancer
What pathway activates CYP1B1?
AhR/RNT pathway
What element is recognized during the activation of CYP1B1?
Dioxin response element (DRE)
What is linked with demethylation of CpG islands in prostate cancer?
Increased levels of CYP1B1
What transcription factor binding site was demethylated in prostate cancer tissues?
Sp1 binding site GGGCGG
What did the study suggest about the methylation of sites in the promoter and enhancer regions of CYP1B1?
Hypomethylation allows binding transcription factors and promotes expression
Who conducted the study referenced regarding methylation and CYP1B1?
Tokizane et al, 2005
