Modular Microfluidic System for Emulation of Human Phase I/Phase II Metabolism

We present a microfluidic device for coupled phase I/phase II metabolic reactions in vitro. The chip consists of microchannels, which are used as packed bed reactor compartments, filled with superparamagnetic microparticles bearing recombinant microsomal phase I cytochrome P450 or phase II conjugating enzymes (UDP-glucuronosyl­transferase). Online coupling of the microfluidic device with LC/MS enabled the quantitative assessment of coupled phase I/phase II transformations, as demonstrated for two different substrates, 7-benzyloxy-4-trifluoro­methyl­coumarin (BFC) and dextromethorphan (DEX). In contrast, conventional sequential one-pot incubations did not generate measurable amounts of phase II metabolites. Because the microfluidic device is readily assembled from standard parts and can be equipped with a variety of recombinant enzymes, it provides a modular platform to emulate and investigate hepatic metabolism processes, with particular potential for targeted small-scale synthesis and identification of metabolites formed by sequential action of specific enzymes

N-acetyltransferase 1*10 genotype in bladder cancer patients

<p>In a large bladder cancer study in the greater Berlin area with 425 cases and 343 controls, the haplotype <i>N-acetyltransferase 1*10</i> (<i>NAT1*10</i>) was associated with a decreased bladder cancer risk. In a recently published meta-analysis, results of the studies were found to be inconclusive. Therefore, the aim of this study was to investigate the frequency of <i>NAT1*10</i> in bladder cancer patients and controls recruited in an area without industries reported to be associated with increased bladder cancer risk. Rs1057126 (1088 T > A) and rs15561 (1095 C > A) were determined in 412 bladder cancer patients and 415 controls without a known history of malignancies. With these two single-nucleotide polymorphisms (SNP), it was possible to distinguish between <i>NAT1*4</i> (wild type), <i>NAT1*3</i> (1095 C > A), and <i>NAT1*10</i> (1088 T > A, 1095C > A). The frequencies of the determined NAT1 haplotypes did not differ markedly between cases and controls: <i>NAT1*4</i>: 74%, <i>NAT1*3</i>: 6%, <i>NAT1*10</i>: 20%. Bladder cancer risk was not significantly modulated by <i>NAT1*10/*10</i> (OR 1.03, 95% CI 0.71–1.48) but was higher for <i>NAT1*3/*3</i> genotypes (OR 2.05, 95% CI 1.32–3.21). In contrast to the Berlin study from 2001, data in present study demonstrated that <i>NAT1*10</i> haplotype was not associated with a significantly decreased bladder cancer risk. This may be due to local effects in the greater Berlin area, particularly at the time of investigation. The findings of the present study are in agreement with observations of a recently published meta-analysis which also showed no relevant impact of <i>NAT1*10</i> haplotype on bladder cancer risk. The impact of the rare <i>NAT1*3/*3</i> genotype was significant but this may be attributed to rarity without major practical relevance.</p

Top ten three-way interactions found in the analysis of the ever smokers.

<p>The top ten of the 1,760 possible three-way interactions comprised of the six SNPs and <i>GSTM1</i>, as well as their odds ratios (OR) with 95% confidence intervals (CI) are listed in order of their p-values, where the p-values were adjusted for multiple comparisons by the Bonferroni correction.</p

Population attributable risks and odds ratios due to genetic factors.

<p>Population attributable risks (PARs) and odds ratios (ORs) were calculated from the data of the present study and summarized from previously published studies for different genetic factors. Numbers in brackets refer to the publications in which the PARs and ORs were published.</p>a<p>Adjusted for age, gender, smoking habits, all measured SNPs and study site; crude PAR/OR: 16%/1.39; adjusted for age and gender: 16%/1.37; adjusted for all measured SNPs: 15%/1.36.</p>b<p>Adjusted for age, gender, smoking habits, all measured SNPs and study site; crude PAR/OR: 5%/1.09; adjusted for age and gender: 3%/1.05; adjusted for all measured SNPs: 5%/1.10.</p>c<p>Data from Moore et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880-Moore1" target="_blank">[48]</a> and Garcia-Closas et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880-GarcaClosas2" target="_blank">[47]</a> result in PARs of 2–18%.</p>d<p>Combined PAR, individual SNP OR and PAR adjusted for age, gender, smoking habits and all measured SNPs.</p>e<p>Range of individual SNP OR adjusted for age, gender, smoking habits, all measured SNPs and study site depending on the mode of inheritance.</p

Optimal odds ratios for combinations of one to seven polymorphisms.

<p>For the computation of the optimal odds ratios (OR), all possible combinations of one to seven of the polymorphisms rs1014971, rs9642880, rs710521, rs8102137, rs11892031, rs1495741 and <i>GSTM1</i> were considered. (A) Profile plots for the odds ratios in the total group (black line) and the subgroups of ever smokers (red line), current smokers (green), former smokers (blue) and non-smokers (cyan). The lines were included for clarity of information and not to suggest a continuous development. Dashed lines indicate when number of cases and/or number of controls fall below 100. In these situations, the corresponding odds ratios should be interpreted with caution. (B)–(F): For the optimal combinations shown in (A), box plots of odds ratios computed in 500 bootstrap samples from (B) the total group, (C) the ever smokers, (D) the current smokers, (E) the former smokers and (F) the non-smokers. In twelve of the bootstrap samples (all but one in the analyses of the seven-way interactions in the total and the smoker group), the odds ratios were larger than 15. For a better presentation, these odds ratios are not displayed in the corresponding box plots. The crosses mark the odds ratios of the optimal combinations in the original analysis. The corresponding plots of the test statistics are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s001" target="_blank">Figure S1</a>.</p

Relative risks and frequency of risk factors assuming a PAR of 30%.

<p>Relative risks are calculated depending on the frequency of the risk factor in the population assuming a population attributable risk (PAR) of 30%, corresponding to the supposed PAR of genetic risk factors for UBC. Given a PAR of 30%, the relative risk does not fall below 1.43 if the frequency of the risk factor is present in almost the entire population.</p

Number of times the considered polymorphisms appear in the ten top two- and three-way interactions when analyzing the different smoker groups.

<p>Numbers in brackets are from the analysis of the two-way interactions. Numbers outside the brackets are from the analysis of the three-way interactions. The corresponding groupwise top ten two-way interactions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s016" target="_blank">Tables S12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s017" target="_blank">S13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s018" target="_blank">S14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s019" target="_blank">S15</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s020" target="_blank">S16</a>, and the top ten three-way interactions are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s021" target="_blank">Tables S17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s022" target="_blank">S18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s023" target="_blank">S19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s024" target="_blank">S20</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051880#pone.0051880.s025" target="_blank">S21</a>.</p

Population attributable risks and odds ratios in the different smoker groups.

<p>Population attributable risks (PARs) and odds ratios (ORs) were calculated from the data of the present study and summarized from previously published studies for the different smoker groups, partly stratified by gender (M: Male, F: Female), where non-smokers were used as a reference group having no additional risk. Numbers in brackets refer to the publications in which the PARs and ORs were published.</p>a<p>Adjusted for age and gender; crude PAR/OR: 39%/2.65; adjusted for age, gender, SNPs: 30%/2.15.</p>b<p>Adjusted for age and gender; crude PAR/OR: 29%/3.21; adjusted for age, gender, SNPs: 28%/3.17.</p>c<p>Adjusted for age and gender; crude PAR/OR: 51%/2.83; adjusted for age, gender, SNPs: 46%/2.47.</p

Top ten two-way interactions found in the analysis of the non-smokers.

<p>The top ten of the 288 possible two-way interactions comprised of the six SNPs and <i>GSTM1</i> as well as their odds ratios (OR) with 95% confidence intervals (CI) are listed in order of their p-values, where the p-values were adjusted for multiple comparisons by the Bonferroni correction.</p

Comparison of epigenetic modifier transcript levels between liver and brain.

<p>(<b>A</b>) Schematic diagram showing sample preparation and analysis of a set of 156 epigenetic modifier genes. (<b>B</b>) Human hepatocytes were stained 24 h after plating with antibodies specific for dipeptidyl peptidase (DPP4) or albumin (ALB). Nuclei were stained with the DNA dye H-33342 (blue). Data are representative for preparations from three different donors. Scale bars: 100 µm. (<b>C</b>) The mRNA was isolated from three preparations of freshly-isolated hepatocytes and analyzed by RT-qPCR for hepatic (ALB, CYP3A, CYP7A1, DPP4, HNF4, MET) and neuronal (TH, DCX, TUBB3) differentiation markers. Gene expression levels are indicated relative to hESC as reference cell line and a set of three reference genes (HPRT, RPL13A, GAPDH) was used for internal calibration. (<b>D</b>) Transcript levels of epigenetic modifiers were measured by RT-qPCR in human cortex (Ctx), liver (huHep) and embryonic stem cells (hESC). Data for Ctx and huHep are indicated as relative change compared to hESC (as reference cell). For comparative display, a scatter plot was constructed so that differentially expressed genes that show pos. association (between Ctx and huHep) are found in red fields, and those that differed in the sense of regulation fall into blue fields. Values of>10 were set to 10. For quadrant count ratio analysis (QCR) only expression values >2 or <−2 were included. (<b>E</b>) The data measured in D were plotted as heat map, sorted according to rel. Ctx expression levels. Transcripts that were >2-fold higher expressed in tissue than in hESC are marked in red, >2-fold lower expression is marked in blue. The color scale ranges from a fold regulation of −20 (dark blue) to +20 (dark red). Measures of variance and p-values are indicated in the supplemental material, genes not regulated significantly (vs. hESC) are displayed as “n.s.”. Specific examples of differential regulation between Ctx and huHep are emphazised by black boxes.</p