Genetic and Nongenetic Factors Associated with Protein Abundance of Flavin-Containing Monooxygenase 3 in Human Liver

Abstract

Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S)-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% (P < 0.001) and 79.5% (P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver.

Fig. 1.

Association between FMO3 protein abundance (pmol/mg protein) and BZD N-oxidation activity in human liver microsomes (n = 37).

Fig. 2.

Association between age and FMO3 protein levels in human liver without (A and B) and with (C and D) considering interplay with diplotype E158K-E308G. (A and C) represent categorical data, and (B and D) show continuous data. Dots indicate individual protein abundance; the mean and S.D. are presented as the horizontal line and error bar. The Kruskal-Wallis test followed by Dunn’s multiple comparison test were used to compare protein levels; ** and *** indicate P values of < 0.01 and < 0.001, respectively.

Fig. 3.

Association between gender and FMO3 protein (A) and mRNA (B) levels in human liver. Dot plots are displayed with mean abundance as the horizontal line, and the error bar displays S.D. with individual maximum and minimum values shown in the dot plot. The Mann-Whitney test was used to compare FMO3 levels; * and *** indicate P values of < 0.05 and < 0.001, respectively.

Fig. 4.

Association between genetic variation and FMO3 protein levels in HLMs (with donor age over 6 years). (A–F) represent individual SNPs (i.e., rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, respectively. Dots indicate individual protein abundance, and the mean and S.D. are presented as the horizontal line and error bar. The Kruskal-Wallis test followed by Dunn’s multiple comparison test were used to compare protein levels; *, ** and *** indicate P values of < 0.05, < 0.01, and < 0.001, respectively.

Fig. 5.

A schematic representation of human FMO3 gene structure and LD generated by Haploview 4.2. The HW P-value cutoff and minimum MAF were 0.01 and 0.02, respectively.

Fig. 6.

Association between FMO3 diplotypes and FMO3 protein levels in human liver (with donor age over 6 years). Only those diplotypes that show statistical significance in FMO3 protein abundance are presented. Dots indicate individual protein abundance, and the mean and S.D. are presented as the horizontal line and error bar. The Kruskal-Wallis test followed by Dunn’s multiple comparison test were used to compare protein levels; *, **, and *** indicate P values of < 0.05, < 0.01 and < 0.001, respectively.