RNA Sequencing Quantification of Xenobiotic-Processing Genes in Various Sections of the Intestine in Comparison to the Liver of Male Mice

Abstract

Previous reports on tissue distribution of xenobiotic-processing genes (XPGs) have limitations, because many non-cytochrome P450 phase I enzymes have not been investigated, and one cannot compare the real mRNA abundance of multiple XPGs using conventional quantification methods. Therefore, this study aimed to quantify and compare the mRNA abundance of all major XPGs in the liver and intestine using RNA sequencing. The mRNA profiles of 304 XPGs, including phase I, phase II enzymes, phase II cosubstrate synthetic enzymes, xenobiotic transporters, as well as xenobiotic-related transcription factors, were systematically examined in the liver and various sections of the intestine in adult male C57BL/6J mice. By two-way hierarchical clustering, over 80% of the XPGs had tissue-divergent expression, which partitioned into liver-predominant, small intestine-predominant, and large intestine-predominant patterns. Among the genes, 54% were expressed highest in the liver, 21% in the duodenum, 4% in the jejunum, 6% in the ileum, and 15% in the large intestine. The highest-expressed XPG in the liver was Mgst1; in the duodenum, Cyp3a11; in the jejunum and ileum, Ces2e; and in the large intestine, Cyp2c55. Interestingly, XPGs in the same family usually exhibited highly different tissue distribution patterns, and many XPGs were almost exclusively expressed in one tissue and minimally expressed in others. In conclusion, the present study is among the first and the most comprehensive investigations of the real mRNA abundance and tissue-divergent expression of all major XPGs in mouse liver and intestine, which aids in understanding the tissue-specific biotransformation and toxicity of drugs and other xenobiotics.

Fig. 1.

Summary of the mRNA expression of all XPGs in the liver and intestine. Liver (liv) and various sections of the intestine [duodenum (duo), jejunum (jej), ileum (ile), and large intestine (LI)] from C57BL/6J male mice ages 2–3 months were used for RNA-Seq quantification. (A) Tissue distribution of cumulative mRNAs of all XPGs and genes in each category (namely, phase I enzymes, phase II enzymes, uptake transporters, efflux transporters, and transcription factors). Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). (B) Percentages of the 304 XPGs that are not expressed, equally expressed, or differentially expressed in the liver and intestine. (C) Two-way hierarchical clustering of the 252 XPGs with differential expression in at least two tissues. The dendrogram describes the relationship between different gene expression profiles and various tissues. Average FPKM values of three mice per tissue are presented by colored squares: red, relatively high expression; blue, relatively low expression. The solid lines separate the expression profiles into three clusters of liver-predominant, small intestine–predominant, and large intestine–predominant expression. (D) Cumulative mRNAs of genes in each cluster in the liver and intestine. (E) Number of XPGs with highest mRNA expression in each tissue.

Fig. 2.

Tissue distribution of Ces (A), Pon (B), and Ephx (C) mRNAs in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). The tissue distribution of cumulative mRNAs of genes in each gene family are shown with a red title. duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.

Fig. 3.

Tissue distribution of Akr (A), Cbr (B), and Nqo (C) mRNAs in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). The tissue distribution of cumulative mRNAs of genes in each gene family is shown with red title. duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.

Fig. 4.

Tissue distribution of some P450 mRNAs (Cyp1a1 to Cyp4a12b) in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). The tissue distribution of cumulative mRNAs of all P450s is shown with a red title. duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.

Fig. 5.

Tissue distribution of other P450 (Cyp4a14 to Cyp4v3) and P450 (cytochrome) oxidoreductase (Por) (A), Adh (B), Aldh and Aox (C), and Fmo (D) mRNAs in liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.

Fig. 6.

Tissue distribution of Ugt (A), Sult (B), and Gst (C) mRNAs in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). The tissue distribution of cumulative mRNAs of genes in each gene family are shown with red title. duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.

Fig. 7.

Tissue distribution of mRNAs of enzymes responsible for methylation, acetylation, and amino acid conjugation (A), and synthesis of cosubstrates for phase II conjugation reactions in the liver and intestine (B). Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). As₃mt, arsenic (+3 oxidation state) methyltransferase; Bal, bile acid–CoA ligase; Bat, bile acid–CoA:amino acid N-acyltransferase; Comt, catechol O-methyltransferase; duo, duodenum; Gclc, glutamate-cysteine ligase catalytic subunit; Gclm, glutamate-cysteine ligase modifier subunit; Glyat, glycine-N-acyltransferase; ile, ileum; jej, jejunum; LI, large intestine; liv, liver; Tpmt, thiopurine S-methyltransferase; Ugdh, UDP-glucose 6-dehydrogenase; Ugp2, UDP-glucose pyrophosphorylase 2.

Fig. 8.

Tissue distributio(n of mRNAs of Abc (A) and Slc xenobiotic transporters (B) in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). The tissue distribution of cumulative mRNAs of all Abc and Slc drug transporters are shown with a red title. Abst, apical sodium-dependent bile acid transporter; Cnt, concentrative nucleoside transporter; duo, duodenum; Ent, equilibrative nucleoside transporter; ile, ileum; jej, jejunum; LI, large intestine; liv, liver; Mdr, multidrug resistance protein; Mrp, multidrug resistance-associated protein; Ntcp, Na⁺-taurocholate cotransporting polypeptide; Oat, organic anion transporter; Oatp, organic anion transporting polypeptide; Oct, organic cation transporter; Octn, organic cation/carnitine transporter.

Fig. 9.

Tissue distribution of mRNAs of genes encoding xenobiotic-related transcription factors in the liver and intestine. Data are presented as the mean FPKM ± S.E.M. of three individual animals. Any groups with the same letter are not different from each other (significance set at p < 0.05, one-way ANOVA followed by Duncan's post-hoc test). duo, duodenum; ile, ileum; jej, jejunum; LI, large intestine; liv, liver.