ABSTRACT:HepaRG cells, derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes. More importantly, differentiated HepaRG cells are able to maintain activities of many xenobiotic-metabolizing enzymes, and expression of the metabolizing enzyme genes can be induced by xenobiotics. The ability of these cells to express and induce xenobiotic-metabolizing enzymes is in stark contrast to the frequently used HepG2 cells. The previous studies have mainly focused on a set of selected genes; therefore, it is of significant interest to know the extent of similarity of gene expression at whole genome levels in HepaRG cells and HepG2 cells compared with primary human hepatocytes and human liver tissues. To accomplish this objective, we used Affymetrix (Santa Clara, CA) U133 Plus 2.0 arrays to characterize the whole genome gene expression profiles in triplicate biological samples from HepG2 cells, HepaRG cells (undifferentiated and differentiated cells), freshly isolated primary human hepatocytes, and frozen liver tissues. After using similarity matrix, principal components, and hierarchical clustering methods, we found that HepaRG cells globally transcribe genes at levels more similar to human primary hepatocytes and human liver tissues than HepG2 cells. In particular, many genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 cells compared with primary human hepatocytes and liver samples. The transcriptomic similarity of HepaRG with primary human hepatocytes is encouraging for use of HepaRG cells in the study of xenobiotic metabolism, hepatotoxicology, and hepatocyte differentiation.

Polymorphisms in the POR gene can affect POR and P450-catalyzed drug oxidation. These results suggest that POR has the potential to serve as a predictive biomarker for pharmacogenomic testing.

This assay can be applied in pharmacogenomic studies in both basic research and clinical laboratories. It is also an ideal technology for pharmacogenomic tests in both developed and developing countries.

Background: Cholesterol 7-alpha-hydroxylase (CYP7A1) is the rate limiting enzyme for converting cholesterol into bile acids. Genetic variations in the CYP7A1 gene have been associated with metabolic disorders of cholesterol and bile acids, including hypercholesterolemia, hypertriglyceridemia, arteriosclerosis, and gallstone disease. Current genetic studies are focused mainly on analysis of a single nucleotide polymorphism (SNP) at A-278C in the promoter region of the CYP7A1 gene. Here we report a genetic approach for an extensive analysis on linkage disequilibrium (LD) blocks and haplotype structures of the entire CYP7A1 gene and its surrounding sequences in Africans, Caucasians, Asians, Mexican-Americans, and African-Americans.

Cytochrome P450 oxidoreductase (POR) is the single flavoprotein which donates electrons to the microsomal cytochrome P450 enzymes for oxidation of their substrates. In this study, we sequenced all 15 exons and the surrounding intronic sequences of POR in 100 human liver samples to identify novel and confirm known genetic polymorphisms in POR. Thirty-four single nucleotide polymorphisms (SNPs) were identified including 9 in the coding exons (5 synonymous and 4 nonsynonymous), 20 in the intronic regions, and 5 in the 3'-UTR. Of these, 9 were novel SNPs, including three nonsynonymous SNPs, SNH313003 (817733G>C; K49N), SNH313020 (848661C>A; L420M), and SNH313029 (849577T>C; L577P) with minor allele frequencies of 0.005, 0.045, and 0.020, respectively. We also confirmed a previously reported non-synonymous SNP rs1057868 (A503V) as well as five synonymous SNPs (G5G, T29T, P129P, S485S, and S572S) all with allele frequencies similar to those previously reported. Structurally, these polymorphisms occur in different regions: SNH313003 (K49N) in the amino-terminal tail, SNH313020 (L420M) in the connecting domain, SNH313029 (L577P) in the NADPH-binding domain, and rs1057868 (A503V) in the FAD binding domain.

Cytochrome P450 oxidoreductase (POR) is the only protein that donates electrons to all microsomal P450 enzymes, which metabolize more than 80% of prescription drugs. Our objective was to determine if polymorphisms in POR could affect gene expression and activity of POR, and/or P450‐catalyzed drug metabolism. POR polymorphisms were identified from non‐diseased human livers (n=99) by sequencing the POR gene and mRNA levels were quantified by the branched DNA method. POR activity was quantified by measuring cytochrome c reduction in liver microsomes and activities of ten drug‐metabolizing P450 enzymes were quantified by HPLC. Of the 34 identified polymorphisms, 9 were novel, 9 were in exons, and 4 changed an amino acid: K49N, L420M, A503V, and L577P. We report a novel exonic polymorphism, L577P (patent pending), which is associated with decreased POR (p=0.003) and several P450 activities. Our data suggest that [1] POR is functionally polymorphic in the general population and [2] has the potential to serve as a biomarker to optimize pharmacologic dosing strategies. This work was supported by NIH COBRE 5P20 RR021940 and Pharmaceutical Research and Manufacturers of America Foundation (PhRMA).

RNA polymerase II core promoter and enhancer/repressor elements are involved in regulation of gene transcription. The objective of this study is to examine whether genetic polymorphisms in these elements can change regulation of gene transcription. Genetic polymorphisms in the UGT1A1 promoter were evaluated, which include polymorphism of (TA)6/7TAA (*1/*28) in the TATA box, and two SNPs (−3279 T>G *60 and .C3156 G>A*93) in the distal enhancer elements. These polymorphisms in 100 human liver samples were genotyped by sequencing or microarray chips. mRNA levels of UGT1A1 and 9 transcriptional factors (PXR, CAR, PPARa, GR, Nrf2, AhR, HNF1a, HFN4a, and CEBPa) were quantified by the bDNA technique. Associations were established by multiple linear regressions with adjustment for confounders. Correlations of gene transcription between UGT1A1 and each of the 9 transcriptional factors were observed in the samples carrying genotypes of *1/*1 and *1/*28. However, such correlations did not exist in the samples carrying combinational genotypes of *28/*28 with *60/*60 or *93/*93, which also associated with significantly lower UGT1A1 mRNA levels. Polymorphisms in both RNA polymerase II core promoter and enhancer elements of the UGT1A1 promoter may have a synergetic effect on regulation of UGT1A1 transcription, but the mechanisms need to be investigated. This work was supported by NIH COBRE 5P20 RR021940.

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