Association Between Polymorphisms In UGT Genes And Metabolism Of Exemestane And NNAL

Phase ? conjugation reactions act as an important process of biotransformation of endogenous compounds and xenobiotics.Major Phase ? enzymes include UDP-glucuronosyltransferases(UGTs),sulfotransferases,N-acetyltransferases,glutathione S-transferases(GSTs)and methyltransferases.UGTs are the key enzymes of the process known as glucuronidation,responsible for the metabolism of many xenobiotics and endobiotics,while the GSTs are enzymes that catalyze the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds.Exemestane(EXE)is an aromatase inhibitor widely used for the prevention and treatment of breast cancer.The major metabolic pathway for EXE is reduction to form the active 17?-dihydro-EXE(17?-DHE)and subsequent glucuronidation to 17?-hydroxy-EXE-17-O-?-D-glucuronide(17?-DHE-Gluc).UGT2B17 is known as the major enzyme responsible for formation of glucuronide of 17?-DHE in vitro.The first aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17?-DHE-Gluc and 17P-DHE in patients taking EXE.Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study.UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE,17?-DHE and 17?-DHE-Gluc were quantified by UPLC/MS in patients' urine and plasma.A 39-fold decrease(P<0.0001)and a 29-fold(P<0.0001)decrease have been confirmed in the levels of creatinine-adjusted urinary and plasma 17?-DHE-Gluc respectively in subjects with 0 copy of UGT2B17 gene(the UGT2B17(*2/*2)genotype)vs subjects with 2 copies of UGT2B17 gene(the UGT2B17(*1/*1)genotype).The results show that UGT2B17 is the major enzyme responsible for 17?-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17?-DHE levels in vivo.Though previous studies have demonstrated that EXE is reduction to form the active 17?-dihydro-EXE(17?-DHE)by carbonyl reductase 1(CBR1)and aldo-keto reductase 1C1(AKR1C1),AKR1C2,AKR1C3,and AKR1C4 and subsequent glucuronidation to 17?-hydroxy-EXE-17-O-?-D-glucuronide(17?-DHE-Gluc)by UGT2B17,the metabolic pathway of EXE has not been fully characterized.The second aim of the present study was to identify new phase ? metabolites in patients taking EXE.An approach named MSE was used for metabolites identification in human urine samples and matched plasma samples.Two new metabolites have been detected and predicted as EXE-cysteine and 17?-DHE-cysteine by elemental composition and mass defect.The cysteine conjugates may be formed during the process of GSH conjugation,and followed by sequential removal of gamma-glutamyl and glycyl moieties from the GSH conjugates.The two novel metabolites of EXE have been confirmed as major metabolites in vivo by quantification via ultra-pressure liquid chromatography-mass spectrometry(UPLC/MS)analysis.Therefore,the functional gene polymorphisms in enzymes responsible for GSH conjugation may affect the metabolism of EXE.The most abundant and potent carcinogenic tobacco-specific nitrosamines in tobacco and tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK).In vivo,NNK is rapidly metabolized to both the(R)-and(S)-enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL),which possesses similar carcinogenic properties as NNK.The major detoxification pathway for both NNAL enantiomers is glucuronidation by UDP-glucuronosyltransferase(UGT)enzymes including UGT2B10 and UGT2B17.The third aim of the present study is to determine the functional polymorphisms in UGT2B17,UGT2B10 and UGT2B7 on the levels of NNAL-N-Gluc and individual NNAL-O-Gluc diastereomers in urine specimens of smokers.NNAL-N-Gluc,(R)-NNAL-O-Gluc,(S)-NNAL-O-Gluc and free NNAL were simultaneously and directly quantified in the urine of smokers by LC/MS analysis.Genotypes were determined by Taqman-assay using genomic DNA.The average percentage of total urinary NNAL that existed as NNAL-N-Gluc,(R)-NNAL-O-Gluc,(S)-NNAL-O-Gluc,and free-NNAL in 180 active smokers was 23.2%,21.7%,26.9%and 28.2%,respectively.The functional knock-out polymorphism in the UGT2B10 gene at codon 67(Asp>Tyr)was significantly(P<0.0001)associated with a 93%decrease in creatinine-adjusted NNAL-N-Gluc.The polymorphic whole-gene deletion of the UGT2B17 gene was associated with significant(P=0.0048)decreases in the levels of creatinine-adjusted(R)-NNAL-O-Gluc,with a 32%decrease in the levels of urinary(R)-NNAL-?-Gluc/(S)-NNAL-?-Gluc among subjects with the UGT2B17(*2/*2)genotype as compared to subjects with the UGT2B17(*1/*1)genotype.These results show that the functional polymorphisms in UGTs are associated with altered detoxification capacity against NNAL and may therefore affect individual cancer risk upon exposure to tobacco.The present studies showed the association between UGTs gene polymorphsims and metabolism of Exemestane and NNAL and suggested UGTs gene polymorphsims may play a role in variability in EXE-induced toxicity and therapeutic efficacy between patients and cancer risk in smokers;identified two new metabilites of EXE for fully characterization metabolism of EXE through MSE,suggested functional polymorphisms in GSTs may affect metabolic pathway of EXE.