DMD#63545 Drug Metabolism & Disposition RNA-Seq Quantification of Hepatic Drug Processing Genes in Germ-Free Mice Felcy Pavithra Selwyn, Julia Yue Cui and Curtis D. Klaassen Supplement Figure-1 1500 Alcohol dehydrogenase CV M GF M 1000 500 mRNA (FKPM) mRNA CV:2.56 GF:2.55 0 Adh1 Adh4 Adh5 Adh7 Adh6-ps1 Supplement Figure 1. Gene expression of Alcohol dehydrogenase. Total RNA was isolated from livers of adult male conventional (CV) and germ-free (GF) C57BL/6 mice (n = 3 per group). The mRNA quantified by RNA-Seq as described in methods. (* indicates differential expression determined using Cuffdiff (FDR-BH<0.05)). Dark blue and red bars represent CV and GF male mice, respectively. Adh- Alcohol dehydrogenase, FPKM- fragments per kilobase of exon per million reads mapped. DMD#63545 Supplement Table 1. Comparision of gene expression of drug processing genes in different strains of GF mice compared to their respective CV mice Gene Expression In GF mice compared to CV mice (approx.) GF IQI mice GF NMRI mice GF C57BL6 mice Gene name (Toda et al) (Bjorkholm et al) (this study) Cyp1a2 50% decrease 51% increase Cyp2a4 86% decrease 340% increase No change Cyp2b9 99% decrease 2325% increase 7457% increase Cyp2b10 57% decrease Cyp2c38 140% increase 74% increase Cyp3a11 90% decrease 87% decrease Cyp3a13 50% decrease No change Cyp3a16 100% decrease 86% decrease Cyp3a25 20% decrease No change Cyp3a41 99% decrease No change Cyp3a44 98% decrease 87% decrease Sult1b1 75% increase 70% increase Sult1c2 80% increase No change Sult1d1 75% decrease 95% increase 68% increase Gstt3 100% increase 67% increase Gstp1 110% decrease 66% decrease Ugt1a1 75% increase No change Oatp1a4 100% decrease No change Oct1 43% decrease No change Ntcp 50% decrease 46% increase Mrp3 52% decrease No change AhR 52% decrease 50% increase CAR 80% decrease 100% increase 50% increase PXR 33% decrease No change 1 DMD#63545 Supplement Table 2. Gene expression of lesser known Phase-1 and Phase-2 drug processing genes in livers of CV and GF mice CV M GF M Ratio Gene Mean SEM Mean SEM (GF/CV) Significant? Function Phase-1 genes Ache 0.147 0.019 0.125 0.008 0.850 NO Acetylcholine esterase Bche 12.264 0.160 15.633 0.824 1.275 NO Butrylcholine estarase Pon1 340.735 29.639 428.092 10.186 1.256 NO Paraoxonases (Lactonases) Pon2 24.695 0.771 19.653 0.201 0.796 NO Paraoxonases (Lactonases) Pon3 27.250 1.536 24.611 1.631 0.903 NO Paraoxonases (Lactonases) Akp3 0.176 0.130 0.097 0.079 0.551 NO Alkaline phosphatase Alpi 0.192 0.087 0.173 0.092 0.903 NO Alkaline phosphatase Alpl 2.989 0.306 4.574 0.353 1.531 YES Alkaline phosphatase Gusb 8.599 0.304 6.504 0.111 0.756 NO Beta-glucuronidase Short chain Sdr9c7 21.109 3.034 8.293 1.022 0.393 YES Dehydrogenase/Reductase Short chain Sdr39u1 10.391 0.301 10.677 0.499 1.028 NO Dehydrogenase/Reductase Short chain Sdr16c5 0.184 0.053 0.057 0.024 0.313 NO Dehydrogenase/Reductase Short chain Sdr42e1 45.950 1.338 46.411 4.082 1.010 NO Dehydrogenase/Reductase Dhrs7b 10.552 0.552 11.716 0.048 1.110 NO Dehydrogenase/reductase Dhrs13 1.766 0.041 1.592 0.034 0.902 NO Dehydrogenase/reductase Dhrs11 16.860 0.439 16.148 0.383 0.958 NO Dehydrogenase/reductase Dhrs7 11.542 1.805 13.523 0.757 1.172 NO Dehydrogenase/reductase Dhrs4 92.353 6.260 85.462 0.994 0.925 NO Dehydrogenase/reductase Dhrs1 70.274 5.336 60.964 1.140 0.868 NO Dehydrogenase/reductase Dhrs9 2.210 0.862 0.422 0.015 0.191 YES Dehydrogenase/reductase Dhrs3 71.496 6.603 70.140 0.849 0.981 NO Dehydrogenase/reductase Dhrsx 7.821 0.578 7.109 0.330 0.909 NO Dehydrogenase/reductase Dihydropyrimidine Dpyd 87.499 3.218 89.004 1.854 1.017 NO Dehydrogenase Dimeric Dihydrodiol Dhdh 43.573 3.664 51.128 2.768 1.173 NO Dehydrogenase Gsr 28.228 1.651 21.605 0.830 0.765 NO Glutathione reductase Txnrd1 42.385 1.755 31.912 0.514 0.753 NO Thioredoxin reductases Txnrd2 15.008 1.024 13.562 0.247 0.904 NO Thioredoxin reductases Txnrd3 2.392 0.206 2.536 0.157 1.060 NO Thioredoxin reductases Cyb5 1133.472 76.707 1445.710 31.431 1.275 NO Cytochrome b5 reductase Cyb5b 120.160 5.739 115.718 2.922 0.963 NO Cytochrome b5 reductase Cyb5d1 7.050 0.076 7.714 0.114 1.094 NO Cytochrome b5 reductase Cyb5d2 4.888 0.264 5.031 0.046 1.029 NO Cytochrome b5 reductase Cyb5r1 4.842 0.735 4.192 0.163 0.866 NO Cytochrome b5 reductase 2 DMD#63545 Cyb5r2 0.043 0.006 0.030 0.009 0.711 NO Cytochrome b5 reductase Cyb5r3 172.160 10.106 175.521 3.991 1.020 NO Cytochrome b5 reductase Cyb5r3 172.160 10.106 175.521 3.991 1.020 NO Cytochrome b5 reductase Cyb5r4 4.916 0.038 4.627 0.136 0.941 NO Cytochrome b5 reductase Cyb5rl 3.661 0.114 3.194 0.152 0.872 NO Cytochrome b5 reductase Molybdenum Hydroxylases Suox 51.178 1.484 57.263 2.573 1.119 NO (Molybdoenzymes) Molybdenum Hydroxylases Mocs1 39.039 3.768 41.839 2.714 1.072 NO (Molybdoenzymes) Molybdenum Hydroxylases Mocs2 40.675 1.025 40.185 0.867 0.988 NO (Molybdoenzymes) Molybdenum Hydroxylases Mocs3 3.283 0.230 2.554 0.178 0.778 NO (Molybdoenzymes) Synthesis of the active Mocos 22.215 1.453 18.332 1.888 0.825 NO molybdenum cofactor Synthesis of the active Gphn 21.102 1.363 23.906 1.012 1.133 NO molybdenum cofactor Xdh 28.400 1.783 33.174 1.291 1.168 NO Xanthine Oxidoreductase Maoa 1.851 0.124 1.378 0.083 0.744 NO Monoamine oxidase Maob 58.714 3.543 69.367 3.369 1.181 NO Monoamine oxidase Rnls 0.343 0.030 0.316 0.045 0.922 NO Amine Oxidases Abp1 0.155 0.084 0.151 0.031 0.974 NO Amine Oxidases Smox 1.594 0.410 1.398 0.069 0.877 NO Amine Oxidases Paox 9.650 0.526 10.671 0.106 1.106 NO Amine Oxidases Aoc3 0.035 0.003 0.060 0.022 1.715 NO Amine Oxidases Aoc2 0.359 0.093 0.393 0.060 1.094 NO Amine Oxidases Lox 0.067 0.003 0.083 0.009 1.237 NO Amine Oxidases Ptgs1 3.956 0.380 3.459 0.100 0.874 NO Prostaglandin synthases Ptgs2 0.005 0.005 0.005 0.005 0.973 NO Prostaglandin synthases Gpx1 1291.270 73.250 1269.677 23.989 0.983 NO Glutathione peroxidases Gpx2 0.120 0.042 0.155 0.025 1.295 NO Glutathione peroxidases Gpx2-ps1 0.004 0.004 0.016 0.016 4.140 NO Glutathione peroxidases Gpx3 2.161 0.079 2.383 0.116 1.103 NO Glutathione peroxidases Gpx4 100.905 5.206 95.558 0.985 0.947 NO Glutathione peroxidases Gpx5 0.021 0.007 0.019 0.010 0.929 NO Glutathione peroxidases Gpx6 0.676 0.186 3.253 0.297 4.816 YES Glutathione peroxidases Gpx7 1.144 0.101 1.117 0.174 0.977 NO Glutathione peroxidases Gpx8 0.651 0.034 0.562 0.084 0.864 NO Glutathione peroxidases Prdx6 153.877 14.727 187.885 0.697 1.221 NO Peroxiredoxin Prdx5 255.425 9.011 230.322 6.735 0.902 NO Peroxiredoxin Prdx3 65.436 1.743 63.795 1.396 0.975 NO Peroxiredoxin Prdx1 358.607 25.439 315.369 1.091 0.879 NO Peroxiredoxin Prdx2 67.925 1.902 66.511 0.519 0.979 NO Peroxiredoxin 3 DMD#63545 Prdx4 64.448 3.372 65.761 2.390 1.020 NO Peroxiredoxin Pepd 20.097 1.011 17.929 0.306 0.892 NO Peptidase D Lap3 137.093 3.246 125.832 0.933 0.918 NO Leucine aminopeptidase 3 Clpb 6.231 0.432 5.916 0.099 0.949 NO Caseinolytic peptidase B Htra1 0.759 0.035 0.659 0.038 0.868 NO Serine peptidase Htra2 6.447 0.155 5.572 0.237 0.864 NO Serine peptidase Htra3 0.365 0.091 0.483 0.061 1.325 NO Serine peptidase Htra4 0.324 0.112 0.717 0.144 2.210 YES Serine peptidase Aadac 321.432 9.162 348.176 12.606 1.083 NO Arylacetamide deacetylase Esd 84.911 3.785 93.134 2.699 1.097 NO Esterase D Biphenyl hydrolase-like serine Bphl 103.110 7.652 124.667 1.336 1.209 NO hydrolase Kynu 55.781 1.583 58.157 0.934 1.043 NO L-Kynurenine hydrolase Cel 0.059 0.054 0.024 0.015 0.405 NO Carboxyl ester lipase Cela1 15.287 2.875 16.091 1.720 1.053 NO Glycosyl hydrolase Cela3b 0.082 0.082 0.057 0.042 0.693 NO Glycosyl hydrolase Cela2a 0.814 0.202 0.991 0.139 1.218 NO Glycosyl hydrolase Aloxe3 0.006 0.003 0.003 0.003 0.469 NO Arachidonate lipoxygenases Alox8 0.000 0.000 0.003 0.003 18.678 NO Arachidonate lipoxygenases Alox12 0.221 0.038 0.083 0.018 0.376 NO Arachidonate lipoxygenases Alox15 0.022 0.002 0.050 0.008 2.228 NO Arachidonate lipoxygenases Alox5ap 2.293 0.377 2.318 0.437 1.011 NO Arachidonate lipoxygenases Alox5 0.029 0.006 0.032 0.009 1.090 NO Arachidonate lipoxygenases Phase 2 genes Comt 466.513 34.645 339.594 5.000 0.728 NO Catechol-o-methyl transferase As3mt 14.003 1.142 17.516 0.177 1.251 NO Arsenic (iii) methyltransferase Gnmt 1211.730 78.767 1105.270 28.557 0.912 NO Glycine N-methyltransferase Hnmt 10.438 1.251 12.554 0.314 1.203 NO Histamine-N-methyltransferase Indolethylamine N- Inmt 364.275 51.455 608.952 58.712 1.672 YES methyltransferase (INMT) Nicotinamide N- Nnmt 65.877 27.873 47.758 4.165 0.725 NO methyltransferase Tpmt 13.383 1.150 16.026 0.429 1.198 NO Thiopurine methyltransferase Guanidinoacetate N- Gamt 100.898 3.461 120.447 1.332 1.194 NO methyltransferase Tst 147.057 7.615 150.440 2.977 1.023 NO Thiosulfate sulfurtransferase Bile acid-CoA:amino acid N- acyltransferase (Amino acid Baat 122.849 5.715 124.694 2.136 1.015 NO conjugation) Acyl-CoA:amino acid N- acyltransferase (Amino acid Acnat1 23.135 0.921 27.112 1.945 1.172 NO conjugation) 4 DMD#63545 Acyl-CoA:amino acid N- acyltransferase (Amino acid Acnat2 6.543 0.471 7.782 0.881 1.189 NO conjugation) Acsbg1 0.042 0.012 0.039 0.002 0.941 NO Acyl-CoA synthetase Chpt1 85.719 11.674 91.075 3.478 1.062 NO Choline phosphotransferases Hypoxanthine-guanine Hprt 37.422 1.225 38.579 1.070 1.031 NO phosphoribosyltransferase Nme1 48.453 3.765 45.791 0.685 0.945 NO Nucleoside diphosphate kinase Nme2 155.564 4.752 130.674 9.125 0.840 NO Nucleoside diphosphate kinase Nme3 18.241 0.997 18.192 0.714 0.997 NO Nucleoside diphosphate kinase Nme4 1.971 0.171 2.159 0.053 1.095 NO Nucleoside diphosphate kinase Nme5 0.066 0.005 0.033 0.020 0.506 NO Nucleoside diphosphate kinase Nme6 12.440 1.943 11.893 0.710 0.956 NO Nucleoside diphosphate kinase Nme7 3.122 0.065 3.360 0.090 1.076 NO Nucleoside diphosphate kinase 5 .