Impact of Genetic Variants in the Nicotine Metabolism Pathway on Nicotine Metabolite Levels in Smokers

Abstract

Background: Nicotine metabolism is a major factor in nicotine dependence, with approximately 70-80% of nicotine metabolized to cotinine in Caucasians. Cotinine formation is catalyzed primarily by CYP2A6, which also converts cotinine to trans-3’-hydroxycotinine (3HC). The goal of the present study was to examine the effects of CYP2A6 deficiency on nicotine metabolism profiles in vivo and the importance of genetic variants in nicotine metabolizing enzyme genes on urinary nicotine metabolites levels. Methods: Urine samples from 722 smokers who participated in the Singapore Chinese Health Study were analyzed using UPLC-MS/MS to detect nicotine and eight of its urinary metabolites, and a total of 58 variants in 12 genes involved in nicotine metabolism were investigated in 475 of these subjects with informative genotyping data. Results: Urine samples stratified by the ratio of 3HC/cotinine exhibited a 7-fold increase in nicotine-N’-oxide, a 6-fold increase in nicotine-Glucuronide, and a 5-fold decrease in 3HC-Glucuronide when comparing the lower vs upper 3HC/cotinine ventiles. Significant (p<0.0001) associations were observed between functional metabolizing enzyme genotypes and levels of various urinary nicotine metabolites, including CYP2A6 genotype and levels of nicotine, nicotine-Glucuronide, nicotine-N’-oxide and 3HC, UGT2B10 genotype and levels of cotinine, nicotine-Glucuronide and cotinine-Glucuronide, UGT2B17 genotype and levels of 3HC-Glucuronide, FMO3 genotype and levels of nicotine-N’-oxide, and CYP2B6 genotype and levels of nicotine-N’-oxide and 4-hydroxy-4-(3-pyridyl)-butanoic acid. Conclusions: These data suggest that several pathways are important in nicotine metabolism. Impact: Genotype differences in several nicotine metabolizing enzyme pathways may potentially lead to differences in nicotine dependence and smoking behavior and cessation

Biochemical and genetic biomarkers associated with nicotine dependence in Mexican smokers

“…16,18 corresponding decrease in 3HC-O-Gluc formation. 19 In the present study, deficiencies in CYP2A6 activity as measured by the NMR resulted in increased levels of nicotine-N′-oxide and decreased levels of 3HC-O-Gluc, particularly in ND subjects. The rs2431413 SNP in CYP2A6 has been associated with decreased enzyme function.…”

“…In contrast, in subjects with deficient CYP2A6 activity, increased levels of nicotine‐N′‐oxide have been found 16,18 . This deficiency causes rerouting of nicotine metabolism to other pathways, including nicotine oxidation and nicotine glucuronidation, with a corresponding decrease in 3HC‐O‐Gluc formation 19 .…”

“…While CYP2A6 has been suggested to play a role in the formation of 4-hydroxy-4-(3-pyridyl)-bu tanoic acid (HPBA), 18 it is presently unclear whether CYP2A6 is the sole enzyme involved in this pathway (Figure 1). 19 and the levels of 3HC-O-Gluc (p < .0001, r = .6835), while a strong negative correlation was observed with nicotine-N′-oxide (p < .0001, r = .6522) in nicotine-dependent subjects. No correlations were observed in non-nicotine-dependent subjects.…”

Inhibition of Nicotine Metabolism by Cannabidiol (CBD) and 7-Hydroxycannabidiol (7-OH-CBD)

“… 16 An initial screening of the inhibition potential of CBD and 7-OH-CBD against individual CYP and FMO enzymes was determined using microsomes (30–100 μg) from CYP- or FMO-overexpressing HEK293 cell lines in reactions containing either 1 or 10 μM CBD or 7-OH-CBD as potential inhibitors, nicotine or cotinine as substrates, 100 mM potassium phosphate buffer (pH 7.4), and 3 mM MgCl 2 in a final reaction volume of 30 μL. The nicotine and cotinine concentrations used in these reactions are described in Table 1 and were at or near their known Michaelis–Menten constant values ( K M ) for a given enzyme (e.g., nicotine to cotinine, 100 μM; cotinine to 3HC, 100 μM; nicotine to nornicotine, 50 μM; nicotine to NOX, 1 mM; and cotinine to COX, 1 mM 14 , 16 , 19 , 30 − 33 ). As CBD exhibits extensive nonspecific binding (70–90%) to protein and labware, low-binding 0.6 mL microcentrifuge tubes were used for all reactions.…”

“…For nicotine metabolite detection, we used a method identical to that described and validated in previous studies. 33 Briefly, nicotine, NOX, Nic-Gluc, HPBA, cotinine, COX, Cot-Gluc, 3HC, and 3HC-Gluc were detected using a UPLC (Waters Acquity; Waters Corp, Milford, MA) coupled to a triple-quadrupole mass spectrometer equipped with a Zspray electrospray ionization interface (Waters Xevo TQD; Waters Corp) by multiple reaction monitoring (MRM). While a nornicotine standard was not available for the present studies, nornicotine was identified using both parent and daughter scans to develop and confirm an MRM method for nornicotine detection.…”